Lettuce named caballero

ABSTRACT

Novel lettuce, such as lettuce designated CABALLERO is disclosed. In some embodiments, the invention relates to the seeds of lettuce CABALLERO, to the plants and plant parts of lettuce CABALLERO, and to methods for producing a lettuce plant by crossing the lettuce CABALLERO with itself or another lettuce plant. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other lettuce plants derived from the lettuce CABALLERO.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture, to new anddistinctive lettuce (Lactuca sativa) cultivars, such as cultivarsdesignated CABALLERO, and to methods of making and using such plants.

BACKGROUND OF THE INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Lettuce is an important and valuable vegetable crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding lettucecultivar that are agronomically sound or unique. The reasons for thisgoal are to maximize the amount of yield produced on the land used aswell as to improve the plant agronomic and horticultural qualities. Toaccomplish this goal, the lettuce breeder must select and developlettuce plants that have the traits that result in superior cultivars.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope.

In various embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

According to the invention, in some embodiments, there is provided anovel lettuce cultivar, designated CABALLERO. This invention thusrelates to the seeds of lettuce cultivar designated CABALLERO, to theplants or parts thereof of lettuce cultivar designated CABALLERO, toplants or parts thereof consisting essentially all of the physiologicaland morphological characteristics of lettuce cultivar designatedCABALLERO or parts thereof, and/or having all the physiological andmorphological characteristics of lettuce cultivar designated CABALLEROand/or having one or more or all of the characteristics of lettucecultivar designated CABALLERO listed in Table 1 including but notlimited to as determined at the 5% significance level when grown in thesame environmental conditions, and/or having one or more of thephysiological and morphological characteristics of lettuce cultivardesignated CABALLERO listed in Table 1 including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions, and/or having all the physiological andmorphological characteristics of lettuce cultivar designated CABALLEROlisted in Table 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditionsand/or having all the physiological and morphological characteristics oflettuce cultivar designated CABALLERO listed in Table 1 when grown inthe same environmental conditions. The invention also relates tovariants, mutants and trivial modifications of the seed or plant oflettuce cultivar designated CABALLERO.

Plant parts of the lettuce cultivar of the present invention are alsoprovided, such as, but not limited to, a head, leaf, flower, cell,pollen or ovule obtained from the plant cultivar. The present inventionprovides heads and/or leaves of the lettuce cultivar of the presentinvention. Such heads and/or leaves or parts thereof could be used asfresh products for consumption or in processes resulting in processedproducts such as food products comprising one or more harvested part ofthe lettuce plant CABALLERO, for example harvested leaves and/or heads.The harvested part or food product can be or can comprise the lettucehead and/or leaves of the lettuce plant CABALLERO or a salad mixturecomprising leaves of the lettuce plant CABALLERO. The food productsmight have undergone one or more processing steps such as, but notlimited to cutting, washing, mixing, etc. All such products are part ofthe present invention. The present invention also provides plant partsor cells, wherein a plant regenerated from said plants parts or cellshas one or more, or essentially all of the phenotypic and morphologicalcharacteristics of the lettuce plant CABALLERO, such as one or more orall of the characteristics of the lettuce plant CABALLERO, listed inTable 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditions. Allsuch products are part of the present invention.

The plants and seeds of the present invention include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act of The United States of America, e.g., avariety that is predominantly derived from lettuce cultivar designatedCABALLERO or from a variety that i) is predominantly derived fromlettuce cultivar designated CABALLERO, while retaining the expression ofthe essential characteristics that result from the genotype orcombination of genotypes of lettuce cultivar designated CABALLERO; ii)is clearly distinguishable from lettuce cultivar designated CABALLERO;and iii) except for differences that result from the act of derivation,conforms to the initial variety in the expression of the essentialcharacteristics that result from the genotype or combination ofgenotypes of the initial variety or cultivar.

In another aspect, the present invention provides regenerable cells. Insome embodiments, the regenerable cells are for use in tissue culture oflettuce cultivar designated CABALLERO. In some embodiments, the tissueculture is capable of regenerating plants consisting essentially all ofthe physiological and morphological characteristics of lettuce cultivardesignated CABALLERO, and/or having all the physiological andmorphological characteristics of lettuce cultivar designated CABALLERO,and/or having the physiological and morphological characteristics oflettuce cultivar designated CABALLERO, and/or having the characteristicsof lettuce cultivar designated CABALLERO. In one embodiment, theregenerated plants have the characteristics of lettuce cultivardesignated CABALLERO listed in Table 1 including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions. In some embodiments, the plant parts and cellsused to produce such tissue cultures will be embryos, meristematiccells, seeds, callus, pollen, leaves, anthers, pistils, roots, roottips, stems, petioles, heads, cotyledons, hypocotyls, ovaries, seedcoat, fruits, endosperm, flowers, axillary buds or the like. Protoplastsproduced from such tissue culture are also included in the presentinvention. The lettuce shoots, roots and whole plants regenerated fromthe tissue culture, as well as the heads and leaves produced by saidregenerated plants are also part of the invention. In some embodiments,the whole plants regenerated from the tissue culture have one, more thanone, or all of the physiological and morphological characteristics oflettuce cultivar designated CABALLERO listed in Table 1, including butnot limited to as determined at the 5% significance level when grown inthe same environmental conditions.

The invention also discloses methods for vegetatively propagating aplant of the present invention. In the present application, vegetativelypropagating can be interchangeably used with vegetative reproduction. Insome embodiments, the methods comprise collecting a part of a lettucecultivar designated CABALLERO and regenerating a plant from said part.In some embodiments, the part can be for example a leaf cutting that isrooted into an appropriate medium according to techniques known by theone skilled in the art. Plants, plant parts and heads thereof producedby such methods are also included in the present invention. In anotheraspect, the plants and heads thereof produced by such methods consistessentially all of the physiological and morphological characteristicsof lettuce cultivar designated CABALLERO, and/or having all thephysiological and morphological characteristics of lettuce cultivardesignated CABALLERO, and/or having the physiological and morphologicalcharacteristics of lettuce cultivar designated CABALLERO, and/or havingthe characteristics of lettuce cultivar designated CABALLERO. In someembodiments, plants produced by such methods consist of one, more thanone, or all physiological and morphological characteristics of lettucecultivar designated CABALLERO listed in Table 1, including but notlimited to as determined at the 5% significance level when grown in thesame environmental conditions.

Further included in the invention are methods for producing heads,leaves and/or seeds from the lettuce cultivar designated CABALLERO. Insome embodiments, the methods comprise growing a lettuce cultivardesignated CABALLERO to produce a lettuce head, lettuce leaves and/orseeds. In some embodiments, the methods further comprise harvesting thelettuce head leaves and/or seeds. Such lettuce heads, leaves and/orseeds are part of the present invention. In some embodiments, thelettuce heads and/or leaves have all the physiological and morphologicalcharacteristics of the lettuce head and/or leaves of lettuce cultivardesignated CABALLERO (e.g. those listed in Table 1) when grown in thesame environmental conditions.

Also included in this invention are methods for producing a lettuceplant. In some embodiments, the lettuce plant is produced by crossingthe lettuce cultivar designated CABALLERO with itself or another lettuceplant. In some embodiments, the other plant can be a lettuce hybrid orline. When crossed with itself, i.e. when CABALLERO is crossed withanother lettuce cultivar CABALLERO, respectively or self-pollinated,lettuce cultivar CABALLERO will be conserved (e.g. as an inbred). Whencrossed with another, different lettuce plant, an F1 hybrid seed isproduced if the different lettuce plant is an inbred and a “three-waycross” seed is produced if the different lettuce plant is a hybrid. SuchF1 hybrid seed and three-way hybrid seeds and plants produced by growingsaid F1 and three-way hybrid seeds are included in the presentinvention. Methods for producing a F1 and three-way hybrid lettuce seedcomprising crossing lettuce cultivar CABALLERO lettuce plant with adifferent lettuce line or hybrid and harvesting the resultant hybridlettuce seed are also part of the invention. The hybrid lettuce seedsproduced by the methods comprising crossing lettuce cultivar CABALLEROlettuce plant with a different lettuce plant and harvesting theresultant hybrid lettuce seed are included in the invention, as areincluded the hybrid lettuce plants or parts thereof and seeds producedby said grown hybrid lettuce plants.

Further included in the invention are methods for producing lettuceseeds and plants made thereof. In some embodiments, the methods compriseself-pollinating the lettuce cultivar CABALLERO and harvesting theresultant seeds. Lettuce seeds produced by such method are also part ofthe invention.

In another embodiment, this invention also relates to methods forproducing other lettuce plants derived from lettuce cultivar CABALLEROand to the lettuce plants derived by the use of those methods.

In some embodiments, such methods for producing a lettuce plant derivedfrom the lettuce cultivar CABALLERO comprise (a) self-pollinating thelettuce cultivar CABALLERO plant at least once to produce a progenyplant derived from lettuce cultivar CABALLERO; In some embodiments, themethods further comprise (b) crossing the progeny plant derived fromlettuce cultivar CABALLERO with itself or a second lettuce plant toproduce a seed of a progeny plant of a subsequent generation; In someembodiments, the methods further comprise (c) growing the progeny plantof the subsequent generation. In some embodiments, the methods furthercomprise (d) crossing the progeny plant of the subsequent generationwith itself or a second lettuce plant to produce a lettuce plant furtherderived from the lettuce cultivar CABALLERO. In further embodiments,steps (b), steps (c) and/or steps (d) are repeated for at least 1, 2, 3,4, 5, 6, 7, 8, or more generations to produce a lettuce plant derivedfrom the lettuce cultivar CABALLERO. In some embodiments, within eachcrossing cycle, the second plant is the same plant as the second plantin the last crossing cycle. In some embodiments, within each crossingcycle, the second plant is different from the second plant in the lastcrossing cycle.

Another method for producing a lettuce plant derived from the varietyCABALLERO, comprises the steps of: (a) crossing the CABALLERO plant witha second lettuce plant to produce a progeny plant derived from lettucecultivar CABALLERO; In some embodiments, the method further comprises(b) crossing the progeny plant derived from lettuce cultivar CABALLEROwith itself or a second lettuce plant to produce a seed of a progenyplant of a subsequent generation; In some embodiments, the methodfurther comprises (c) growing the progeny plant of the subsequentgeneration; In some embodiments, the method further comprises (d)crossing the progeny plant of the subsequent generation with itself or asecond lettuce plant to produce a lettuce plant derived from CABALLERO.In a further embodiment, step (b), step (c) and/or step (d) are repeatedfor at least 1, 2, 3, 4, 5, 6, 7, 8, or more generation to produce alettuce plant derived from CABALLERO. In some embodiments, within eachcrossing cycle, the second plant is the same plant as the second plantin the last crossing cycle. In some embodiments, within each crossingcycle, the second plant is different from the second plant in the lastcrossing cycle.

In another aspect, the present invention provides methods of introducingor modifying one or more desired trait(s) into the lettuce cultivarCABALLERO and plants or seeds obtained from such methods. The desiredtrait(s) may be, but not exclusively, a single gene. In someembodiments, the gene is a dominant allele. In some embodiments, thegene is a partially dominant allele. In some embodiments, the gene is arecessive allele. In some embodiments, the gene or genes will confersuch traits, including but not limited to male sterility, herbicideresistance, insect resistance, resistance for bacterial, fungal,mycoplasma or viral disease, enhanced plant quality such as improveddrought or salt tolerance, water-stress tolerance, improvedstandability, enhanced plant vigor, improved shelf life, delayedsenescence or controlled ripening, enhanced nutritional quality such asincreased sugar content or increased sweetness, increased texture,flavor and aroma, improved fruit length and/or size, protection forcolor, fruit shape, uniformity, length or diameter, refinement or depth,yield and recovery, improve fresh cut application, specific aromaticcompounds, specific volatiles, leaf texture, specific nutritionalcomponents. For the present invention and the skilled artisan, diseaseis understood to include, but not limited to fungal diseases, viraldiseases, bacterial diseases, mycoplasma diseases, or other plantpathogenic diseases and a disease resistant plant will encompass a plantresistant to fungal, viral, bacterial, mycoplasma, and other plantpathogens. The gene or genes may be naturally occurring lettuce gene(s),mutant(s) or genes modified through the use of New Breeding Techniques.In some embodiments, the method for introducing the desired trait(s) isa backcrossing process making use of a series of backcrosses to lettucecultivar CABALLERO during which the desired trait(s) is maintained byselection. The single gene conversion plants that can be obtained by themethods are included in the present invention.

When dealing with a gene that has been modified, for example through NewBreeding Techniques, the trait (genetic modification) could be directlymodified into the newly developed line/cultivar such as lettuce cultivarCABALLERO. Alternatively, if the trait is not modified into each newlydeveloped line/cultivar such as lettuce cultivar CABALLERO, anothertypical method used by breeders of ordinary skill in the art toincorporate the modified gene is to take a line already carrying themodified gene and to use such line as a donor line to transfer themodified gene into the newly developed line. The same would apply for anaturally occurring trait or one arising from spontaneous or inducedmutations.

In some embodiments, the backcross breeding process of lettuce cultivarCABALLERO comprises (a) crossing lettuce cultivar CABALLERO with plantsthat comprise the desired trait(s) to produce F1 progeny plants. In someembodiments, the process further comprises (b) selecting the F1 progenyplants that have the desired trait(s); In some embodiments, the processfurther comprises (c) crossing the selected F1 progeny plants with thelettuce cultivar CABALLERO plants to produce backcross progeny plants;In some embodiments, the process further comprises (d) selecting forbackcross progeny plants that have the desired trait(s) andphysiological and morphological characteristics of the lettuce cultivarCABALLERO to produce selected backcross progeny plants; In someembodiments, the process further comprises (e) repeating steps (c) and(d) one, two, three, four, five six, seven, eight, nine or more times insuccession to produce selected, second, third, fourth, fifth, sixth,seventh, eighth, ninth or higher backcross progeny plants that have thedesired trait(s) and otherwise consist essentially of all thephysiological and morphological characteristics of the lettuce cultivarCABALLERO, and/or have the desired trait(s) and otherwise all thephysiological and morphological characteristics of the lettuce cultivarCABALLERO, and/or have all the desired trait(s) and otherwise thephysiological and morphological characteristics of the lettuce cultivarCABALLERO as determined in Table 1, including but not limited to whengrown in the same environmental conditions or including but not limitedto at a 5% significance level when grown in the same environmentalconditions. The lettuce plants or seed produced by the methods are alsopart of the invention. Backcrossing breeding methods, well known to oneskilled in the art of plant breeding will be further developed insubsequent parts of the specification.

In an embodiment of this invention is a method of making a backcrossconversion of lettuce cultivar CABALLERO. In some embodiments, themethod comprises crossing lettuce cultivar CABALLERO with a donor plantcomprising a mutant gene(s), a naturally occurring gene(s) or a gene(s)and/or sequences modified through New Breeding Techniques conferring oneor more desired trait to produce F1 progeny plants. In some embodiments,the method further comprises selecting the F1 progeny plant comprisingthe naturally occurring gene(s) mutant gene(s) or modified gene(s)and/or sequences conferring the one or more desired trait. In someembodiments, the method further comprises backcrossing the selectedprogeny plant to the lettuce cultivar CABALLERO. This method may furthercomprise the step of obtaining a molecular marker profile of the lettucecultivar CABALLERO and using the molecular marker profile to select forthe progeny plant with the desired trait and the molecular markerprofile of the lettuce cultivar CABALLERO. The plants or parts thereofproduced by such methods are also part of the present invention.

In some embodiments of the invention, the number of loci that may bebackcrossed into the lettuce cultivar CABALLERO is at least 1, 2, 3, 4,5 or more. A single locus may contain several genes. A single locusconversion also allows for making one or more site specific changes tothe plant genome, such as, without limitation, one or more nucleotidechange, deletion, insertions, etc. In some embodiments, the single locusconversion is performed by genome editing, a.k.a. genome editing withengineered nucleases (GEEN). In some embodiments, the genome editingcomprises using one or more engineered nucleases. In some embodiments,the engineered nucleases include, but are not limited to Zinc fingernucleases (ZFNs), Transcription Activator-Like Effector Nucleases(TALENs), the CRISPR/Cas system, and engineered meganucleasere-engineered homing endonucleases, and endonucleases for DNA guidedgenome editing (Gao et al., Nature Biotechnology (2016), doi:10.1038/nbt.3547). In some embodiments, the single locus conversionchanges one or several nucleotides of the plant genome. Such genomeediting techniques are some of the techniques now known by the personskilled in the art and herein are collectively referred to as “NewBreeding Techniques”. In some embodiments, one or more above-mentionedgenome editing method is directly applied on a plant of the presentinvention, Accordingly, a cell containing edited genome, or a plant partcontaining such cell can be isolated and used to regenerate a novelplant which has a new trait conferred by said genome editing, andotherwise all of the physiological and morphological characteristics oflettuce cultivar CABALLERO.

The invention further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding but not limited to, recurrent selection, backcrossing,pedigree breeding, genomic selection, molecular marker (IsozymeElectrophoresis, Restriction Fragment Length Polymorphisms (RFLPs),Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), AmplifiedFragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, Single NucleotidePolymorphism (SNP), etc.) enhanced selection, genetic marker enhancedselection and transformation. Seeds, lettuce plants, and parts thereofproduced by such breeding methods are also part of the invention.

The invention also relates to variants, mutants and trivialmodifications of the seed or plant of the lettuce cultivar CABALLERO.Variants, mutants and trivial modifications of the seed or plant oflettuce cultivar CABALLERO can be generated by methods available to oneskilled in the art, including but not limited to, mutagenesis (e.g.,chemical mutagenesis, radiation mutagenesis, transposon mutagenesis,insertional mutagenesis, signature tagged mutagenesis, site-directedmutagenesis, and natural mutagenesis), knock-outs/knock-ins, antisenseand RNA interference and other techniques such as the New BreedingTechniques. For more information of mutagenesis in plants, such asagents, protocols, see Acquaah et al. (Principles of plant genetics andbreeding, Wiley-Blackwell, 2007, ISBN 1405136464, 9781405136464, whichis herein incorporated by reference in its entity).

The invention also relates to a mutagenized population of the lettucecultivar CABALLERO and methods of using such populations. In someembodiments, the mutagenized population can be used in screening for newlettuce plants which comprise one or more or all of the morphologicaland physiological characteristics of lettuce cultivar CABALLERO. In someembodiments, the new lettuce plants obtained from the screening processcomprise all of the morphological and physiological characteristics ofthe lettuce cultivar CABALLERO, and one or more additional or differentmorphological and physiological characteristics that lettuce cultivarCABALLERO does not have.

This invention also is directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant wherein either the first or second parent lettuce plant is alettuce cultivar CABALLERO. Further, both first and second parentlettuce plants can come from the lettuce cultivar CABALLERO. Further,the lettuce cultivar CABALLERO can be self-pollinated i.e. the pollen ofa lettuce cultivar CABALLERO can pollinate the ovule of the same lettucecultivar CABALLERO, respectively. When crossed with another lettuceplant, a hybrid seed is produced. Such methods of hybridization andself-pollination are well known to those skilled in the art of breeding.

A lettuce cultivar such as lettuce cultivar CABALLERO has been producedthrough several cycles of self-pollination and is therefore to beconsidered as a homozygous plant or line. An inbred line can also beproduced though the dihaploid system which involves doubling thechromosomes from a haploid plant or embryo thus resulting in an inbredline that is genetically stable (homozygous) and can be reproducedwithout altering the inbred line: Haploid plants could be obtained fromhaploid embryos that might be produced from microspores, pollen, anthercultures or ovary cultures or spontaneous haploidy. The haploid embryosmay then be doubled by chemical treatments such as by colchicine or bedoubled autonomously. The haploid embryos may also be grown into haploidplants and treated to induce the chromosome doubling. In either case,fertile homozygous plants are obtained. A hybrid variety is classicallycreated through the fertilization of an ovule from an inbred parentalline by the pollen of another, different inbred parental line. Due tothe homozygous state of the inbred line, the produced gametes carry acopy of each parental chromosome. As both the ovule and the pollen bringa copy of the arrangement and organization of the genes present in theparental lines, the genome of each parental line is present in theresulting F1 hybrid, theoretically in the arrangement and organizationcreated by the plant breeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross shall be stable. The F1 hybrid is then acombination of phenotypic characteristics issued from two arrangementand organization of genes, both created by a person skilled in the artthrough the breeding process.

Still further, this invention also is directed to methods for producinga lettuce cultivar CABALLERO-derived lettuce plant by crossing lettucecultivar CABALLERO with a second lettuce plant. In some embodiments, themethods further comprise obtaining a progeny seed from the cross. Insome embodiments, the methods further comprise growing the progeny seed,and possibly repeating the crossing and growing steps with the lettucecultivar CABALLERO-derived plant from 0 to 7, or more times. Thus, anysuch methods using the lettuce cultivar CABALLERO are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using lettuce cultivarCABALLERO as a parent are within the scope of this invention, includingplants derived from lettuce cultivar CABALLERO. In some embodiments,such plants have one, more than one, or all physiological andmorphological characteristics of lettuce cultivar designated CABALLEROlisted in Table 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditions. Insome embodiments, such plants might exhibit additional and desiredcharacteristics or traits such as high seed yield, high seedgermination, seedling vigor, early maturity, high yield, diseasetolerance or resistance, and adaptability for soil and climateconditions. Consumer-driven traits, such as a preference for a givenhead and or leaf size, shape, color, texture, taste, are other traitsthat may be incorporated into new lettuce plants developed by thisinvention.

A lettuce plant can also be propagated vegetatively. A part of theplant, for example a shoot or a leaf tissue, is collected, and a newplant is obtained from the part. Such part typically comprises an apicalmeristem of the plant. The collected part is transferred to a mediumallowing development of a plantlet, including for example rooting ordevelopment of shoots. This is achieved using methods well-known in theart. Accordingly, in one embodiment, a method of vegetativelypropagating a plant of the present invention comprises collecting a partof a plant according to the present invention, e.g. a shoot tissue, andobtaining a plantlet from said part. In one embodiment, a method ofvegetatively propagating a plant of the present invention comprises: a)collecting tissue of a plant of the present invention; b) rooting saidproliferated shoots to obtain rooted plantlets. In one embodiment, amethod of vegetatively propagating a plant of the present inventioncomprises: a) collecting tissue of a plant of the present invention; b)cultivating said tissue to obtain proliferated shoots; c) rooting saidproliferated shoots to obtain rooted plantlets. In one embodiment, suchmethod further comprises growing a plant from said plantlets. In oneembodiment, a head is harvested from said plant. In one embodiment, aleaf is harvested from said plant. In one embodiments, such plants,heads and leaves have all the physiological and morphologicalcharacteristics of plants, heads and leaves of lettuce cultivarCABALLERO when grown in the same environmental conditions. In oneembodiment, the head is processed into products prepared cut heads andleaves.

In some embodiments, the present invention teaches a seed of lettucecultivar CABALLERO, wherein a representative sample of seed of saidlettuce cultivar is deposited under NCIMB No. ______.

In some embodiments, the present invention teaches a lettuce plant, or apart thereof, produced by growing the deposited CABALLERO seed.

In some embodiments, the present invention teaches lettuce plant parts,wherein the lettuce part is selected from the group consisting of: aleaf, a flower, a head, an ovule, pollen, and a cell.

In some embodiments, the present invention teaches a lettuce plant, or apart thereof, having all of the characteristics of lettuce cultivarCABALLERO as listed in Table 1 of this application including but notlimited to when grown in the same environmental conditions.

In some embodiments, the present invention teaches a lettuce plant, or apart thereof, having all of the physiological and morphologicalcharacteristics of lettuce cultivar CABALLERO, wherein a representativesample of seed of said lettuce plant was deposited under NCIMB No.______.

In some embodiments, the present invention teaches a tissue culture ofregenerable cells produced from the plant or plant part grown from thedeposited lettuce cultivar CABALLERO seed, wherein cells of the tissueculture are produced from a plant part selected from the groupconsisting of protoplasts, embryos, meristematic cells, callus, pollen,ovules, flowers, seeds, leaves, roots, root tips, anthers, stems,petioles, head, axillary buds, cotyledons and hypocotyls. In someembodiments, the plant part includes protoplasts produced from a plantgrown from the deposited lettuce cultivar CABALLERO seed.

In some embodiments, the present invention teaches a compositioncomprising regenerable cells produced from the plant or plant part grownfrom the deposited lettuce cultivar CABALLERO seed, or other plant partor plant cell. In some embodiments, the composition comprises a growthmedia. In some embodiments, the growth media is solid or a syntheticcultivation medium. In some embodiments, the composition is a lettuceplant regenerated from the tissue culture from a plant grown from thedeposited lettuce cultivar CABALLERO seed, said plant having thecharacteristics of lettuce cultivar CABALLERO, wherein a representativesample of seed of said lettuce cultivar CABALLERO is deposited underNCIMB No. ______.

In some embodiments, the present invention teaches a lettuce headproduced from plant grown from the deposited lettuce cultivar CABALLEROseed.

In one embodiment, the present invention teaches a lettuce leaf producedfrom plants grown from the deposited lettuce cultivar CABALLERO seed.

In some embodiments, the methods of producing said lettuce head comprisea) growing the lettuce plant from deposited lettuce cultivar CABALLEROseed to produce a lettuce head, and b) harvesting said lettuce head. Insome embodiments, the present invention also teaches a lettuce headproduced by the method of producing lettuce head as described above. Inone embodiment, such heads have all the physiological and morphologicalcharacteristics of heads of lettuce cultivar CABALLERO (e.g. thoselisted in Table 1) when grown in the same environmental conditions.

In some embodiments, the methods of producing said lettuce leaf comprisea) growing the lettuce plant from deposited lettuce cultivar CABALLEROseed to produce a lettuce leaf, and b) harvesting said lettuce leaf. Insome embodiments, the present invention also teaches a lettuce leafproduced by the method of producing lettuce leaf as described above. Inone embodiment, such leaves have all the physiological and morphologicalcharacteristics of leaves of lettuce cultivar CABALLERO (e.g. thoselisted in Table 1) when grown in the same environmental conditions.

In some embodiments, the present invention teaches methods for producinga lettuce seed comprising crossing a first parent lettuce plant with asecond parent lettuce plant and harvesting the resultant lettuce seed,wherein said first parent lettuce plant and/or second parent lettuceplant is the lettuce plant produced from the deposited lettuce cultivarCABALLERO seed, or a lettuce plant having all of the characteristics oflettuce cultivar CABALLERO as listed in Table 1 including but notlimited to when grown in the same environmental conditions.

In some embodiments, the present invention teaches methods for producinga lettuce seed comprising self-pollinating the lettuce plant grown fromthe deposited lettuce cultivar CABALLERO seed and harvesting theresultant lettuce seed.

In some embodiments, the present invention teaches the seed produced byany of the above described methods.

In some embodiments, the present invention teaches methods ofvegetatively propagating the lettuce plant grown from the depositedlettuce cultivar CABALLERO seed, said method comprising a) collectingpart of a plant grown from the deposited lettuce cultivar CABALLERO seedand b) regenerating a plant from said part.

In some embodiments, the method further comprises harvesting a head fromsaid vegetatively propagated plant.

In some embodiments, the present invention teaches the plant, the headand leaves thereof of plants vegetatively propagated from plant parts ofplants grown from the deposited lettuce cultivar CABALLERO seed. In oneembodiments, such plant, heads and/or leaves have all the physiologicaland morphological characteristics of lettuce cultivar CABALLERO plant,head and/or leaves of lettuce cultivar CABALLERO (e.g. those listed inTable 1) when grown in the same environmental conditions.

In some embodiments, the present invention teaches methods of producinga lettuce plant derived from the lettuce cultivar CABALLERO. In someembodiment the methods comprise (a) self-pollinating the plant grownfrom the deposited lettuce cultivar CABALLERO seed at least once toproduce a progeny plant derived from lettuce cultivar CABALLERO. In someembodiments, the method further comprises (b) crossing the progeny plantderived from lettuce cultivar CABALLERO with itself or a second lettuceplant to produce a seed of a progeny plant of a subsequent generationand; (c) growing the progeny plant of the subsequent generation from theseed, and crossing the progeny plant of the subsequent generation withitself or a second lettuce plant to produce a lettuce plant derived fromthe lettuce cultivar CABALLERO. In some embodiments said methods furthercomprise the step of: (d) repeating steps (b) and/or (c) for at least 1,2, 3, 4, 5, 6, 7 or more generation to produce a lettuce plant derivedfrom the lettuce cultivar CABALLERO.

In some embodiments, the present invention teaches methods of producinga lettuce plant derived from the lettuce cultivar CABALLERO, the methodscomprising (a) crossing the plant grown from the deposited lettucecultivar CABALLERO seed with a second lettuce plant to produce a progenyplant derived from the lettuce cultivar CABALLERO. In some embodiments,the method further comprises (b) crossing the progeny plant derived fromthe lettuce cultivar CABALLERO with itself or a second lettuce plant toproduce a seed of a progeny plant of a subsequent generation and; (c)growing the progeny plant of the subsequent generation from the seed;(d) crossing the progeny plant of the subsequent generation with itselfor a second lettuce plant to produce a lettuce plant derived from thelettuce cultivar CABALLERO. In some embodiments said methods furthercomprise the steps of: (e) repeating step (b), (c) and/or (d) for atleast 1, 2, 3, 4, 5, 6, 7 or more generation to produce a lettuce plantderived from the lettuce cultivar CABALLERO.

In some embodiments, the present invention teaches plants grown from thedeposited lettuce cultivar CABALLERO seed wherein said plants comprise asingle locus conversion. As used herein, the term “a” or “an” refers toone or more of that entity; for example, “a single locus conversion”refers to one or more single locus conversions or at least one singlelocus conversion. As such, the terms “a” (or “an”), “one or more” and“at least one” are used interchangeably herein. In addition, referenceto “an element” by the indefinite article “a” or “an” does not excludethe possibility that more than one of the elements are present, unlessthe context clearly requires that there is one and only one of theelements. In some embodiments said single locus conversion confers saidplants with a trait selected from the group consisting of malesterility, male fertility, herbicide resistance, insect resistance,resistance for bacterial, fungal, mycoplasma or viral disease, enhancedplant quality such as improved drought or salt tolerance, water stresstolerance, improved standability, enhanced plant vigor, improved shelflife, delayed senescence or controlled ripening, increased nutritionalquality such as increased sugar content or increased sweetness,increased texture, flavor and aroma, improved fruit length and/or size,protection for color, fruit shape, uniformity, length or diameter,refinement or depth lodging resistance, yield and recovery when comparedto a suitable check plant. In some embodiments, the check plant is alettuce cultivar CABALLERO not having said single locus conversion. Insome embodiments, the at least one single locus conversion is anartificially mutated gene or a gene or nucleotide sequence modifiedthrough the use of New Breeding Techniques.

In some embodiments, the present invention provides a method ofproducing a commodity plant product comprising collecting the commodityplant product from the plant of the present invention. The commodityplant product produced by said method is also part of the presentinvention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Adaptability. A plant that has adaptability is a plant able to grow wellin different growing conditions (climate, soils, etc.).

Allele. An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Commodity plant product. A “commodity plant product” refers to anycomposition or product that is comprised of material derived from aplant, seed, plant cell, or plant part of the present invention.Commodity plant products may be sold to consumers and can be viable ornonviable. Nonviable commodity products include but are not limited tononviable seeds and grains; processed seeds, seed parts, and plantparts; dehydrated plant tissue, frozen plant tissue, and processed planttissue; seeds and plant parts processed for animal feed for terrestrialand/or aquatic animal consumption, oil, meal, flour, flakes, bran,fiber, paper, tea, coffee, silage, crushed of whole grain, and any otherfood for human or animal consumption; and biomasses and fuel products;and raw material in industry.

Collection of seeds. In the context of the present invention acollection of seeds is a grouping of seeds mainly containing similarkind of seeds, for example hybrid seeds having the inbred line of theinvention as a parental line, but that may also contain, mixed togetherwith this first kind of seeds, a second, different kind of seeds, of oneof the inbred parent lines, for example the inbred line of the presentinvention. A commercial bag of hybrid seeds having the inbred line ofthe invention as a parental line and containing also the inbred lineseeds of the invention would be, for example such a collection of seeds.

Decreased vigor. A plant having a decreased vigor in the presentinvention is a plant that, compared to other plants has a less vigorousappearance for vegetative and/or reproductive characteristics includingshorter plant height, small fruit size, fewer fruit or othercharacteristics.

Earliness. The earliness relates the number of days from seeding toharvest.

Big Vein. Big vein is a disease of lettuce caused by Mirafiori lettuceBig Vein Virus (MiLBVV, genus Ophiovirus) which is transmitted by thefungus Olpidium virulentus, with vein clearing and leaf shrinkageresulting in plants of poor quality and reduced marketable value.

Bolting. The premature development of a flowering stalk, and subsequentseed, before a plant produces a food crop. Bolting is typically causedby late planting when temperatures are low enough to cause vernalizationof the plants

Core length. The core length is the length of the internal lettuce stemmeasured from the base of the cut and trimmed head to the tip of thestem.

Core diameter: Core diameter is the diameter of the internal leaf stemmeasured at the base of the head.

Corky root. Corky root is a disease caused by the bacterium Sphingomonassuberifaciens, which causes the entire taproot to become brown, severelycracked, and non-functional.

Cupping. In romaine lettuce, cupping is the process by which the romaineforms a heart. Leaves of similar size are formed in the center of thehead and then, the tops of the leaves fold downwards slightly to form aheart.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date

Frame diameter: For frame diameter in case of romaine lettuce, themeasurement is taken from the outer most leaf tip horizontally to theouter most leaf tip. In case of icebergs, frame diameter is measuredwith the outer leaves intact

Head diameter. Diameter of the cut and trimmed head, sliced vertically,and measured at the widest point perpendicular to the stem in case ofromaine lettuce. In case of icebergs, head diameter is measured afterremoving the outer leaves (just the round head).

Head height. Height of the cut and trimmed head, sliced vertically, andmeasured from the base of the cut stem to the cap leaf.

Head weight. Weight of saleable lettuce head, cut and trimmed to marketspecifications.

Immunity to disease(s) and or insect(s). A lettuce plant which is notsubject to attack or infection by specific disease(s) and or insect(s)is considered immune.

Intermediate resistance to disease(s) and or insect(s). A lettuce plantthat restricts the growth and development of specific disease(s) and orinsect(s), but may exhibit a greater range of symptoms or damagecompared to resistant plants. Intermediate resistant plants will usuallyshow less severe symptoms or damage than susceptible plant varietieswhen grown under similar environmental conditions and/or specificdisease(s) and or insect(s) pressure, but may have heavy damage underheavy pressure. Intermediate resistant lettuce plants are not immune tothe disease(s) and or insect(s).

Lettuce Mosaic virus. A disease that can cause a stunted, deformed, ormottled pattern in young lettuce and yellow, twisted, and deformedleaves in older lettuce.

Lettuce Yield (Tons/Acre). The yield in tons/acre is the actual yield ofthe lettuce at harvest.

Maturity (Date). Maturity refers to the stage when plants are of fullsize or optimum weight, and in marketable form or shape to be ofcommercial or economic value. In leaf types they range from 50-75 daysfrom time of seeding, depending upon the season of the year. In othertypes, they range from 65-105 days from time of seeding, depending uponthe season of the year

Nasonovia ribisnigri. A lettuce aphid that colonizes the innermostleaves of the lettuce plant, contaminating areas that cannot be treatedeasily with insecticides.

New Breeding Techniques: New breeding techniques (NBTs) are said ofvarious new technologies developed and/or used to create newcharacteristics in plants through genetic variation, the aim beingtargeted mutagenesis, targeted introduction of new genes or genesilencing (RdDM). The following breeding techniques are within the scopeof NBTs: targeted sequence changes facilitated thru the use of Zincfinger nuclease (ZFN) technology (ZFN-1, ZFN-2 and ZFN-3, see U.S. Pat.No. 9,145,565, incorporated by reference in its entirety),Oligonucleotide directed mutagenesis (ODM, a.k.a., site-directedmutagenesis), Cisgenesis and intragenesis, epigenetic approaches such asRNA-dependent DNA methylation (RdDM, which does not necessarily changenucleotide sequence but can change the biological activity of thesequence), Grafting (on GM rootstock), Reverse breeding,Agro-infiltration for transient gene expression (agro-infiltration“sensu stricto”, agro-inoculation, floral dip), TranscriptionActivator-Like Effector Nucleases (TALENs, see U.S. Pat. Nos. 8,586,363and 9,181,535, incorporated by reference in their entireties), theCRISPR/Cas system (see U.S. Pat. Nos. 8,697,359; 8,771,945; 8,795,965;8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814;8,945,839; 8,993,233; and 8,999,641, which are all hereby incorporatedby reference), engineered meganuclease re-engineered homingendonucleases, DNA guided genome editing (Gao et al., NatureBiotechnology (2016), doi: 10.1038/nbt.3547, incorporated by referencein its entirety), and Synthetic genomics). A major part of today'stargeted genome editing, another designation for New BreedingTechniques, is the applications to induce a DNA double strand break(DSB) at a selected location in the genome where the modification isintended. Directed repair of the DSB allows for targeted genome editing.Such applications can be utilized to generate mutations (e.g., targetedmutations or precise native gene editing) as well as precise insertionof genes (e.g., cisgenes, intragenes, or transgenes). The applicationsleading to mutations are often identified as site-directed nuclease(SDN) technology, such as SDN1, SDN2 and SDN3. For SDN1, the outcome isa targeted, non-specific genetic deletion mutation: the position of theDNA DSB is precisely selected, but the DNA repair by the host cell israndom and results in small nucleotide deletions, additions orsubstitutions. For SDN2, a SDN is used to generate a targeted DSB and aDNA repair template (a short DNA sequence identical to the targeted DSBDNA sequence except for one or a few nucleotide changes) is used torepair the DSB: this results in a targeted and predetermined pointmutation in the desired gene of interest. As to the SDN3, the SDN isused along with a DNA repair template that contains new DNA sequence(e.g. gene). The outcome of the technology would be the integration ofthat DNA sequence into the plant genome. The most likely applicationillustrating the use of SDN3 would be the insertion of cisgenic,intragenic, or transgenic expression cassettes at a selected genomelocation. A complete description of each of these techniques can befound in the report made by the Joint Research Center (JRC) Institutefor Prospective Technological Studies of the European Commission in 2011and titled “New plant breeding techniques—State-of-the-art and prospectsfor commercial development”, which is incorporated by reference in itsentirety.

Plant adaptability. A plant having good plant adaptability means a plantthat will perform well in different growing conditions and seasons.

Plant cell. As used herein, the term “plant cell” includes plant cellswhether isolated, in tissue culture or incorporated in a plant or plantpart.

Plant Part. As used herein, the term “plant part” includes plant cells,plant protoplasts, plant cell tissue cultures from which lettuce plantscan be regenerated, plant calli, plant clumps and plant cells that areintact in plants or parts of plants, such as embryos, pollen, ovules,flowers, seeds, heads, rootstock, scions, stems, roots, anthers,pistils, root tips, leaves, meristematic cells, axillary buds,hypocotyls cotyledons, ovaries, seed coat endosperm and the like. Insome embodiments, the plant part at least comprises at least one cell ofsaid plant. In some embodiments, the plant part is further defined as apollen, a meristem, a cell or an ovule. In some embodiments, a plantregenerated from the plant part has all of the phenotypic andmorphological characteristics of a lettuce of the present invention,including but not limited to as determined at the 5% significance levelwhen grown in the same environmental conditions.

Quantitative Trait Loci (QTL) Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Ratio of head height/diameter. The ratio is the head height divided bythe head diameter and is an indication of the head shape; <1 isflattened, 1=round, and >1 is pointed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Resistance to disease(s) and or insect(s). A lettuce plant thatrestricts the growth and development of specific disease(s) and orinsect(s) under normal disease(s) and or insect(s) attack pressure whencompared to susceptible plants. These lettuce plants can exhibit somesymptoms or damage under heavy disease(s) and or insect(s) pressure.Resistant lettuce plants are not immune to the disease(s) and orinsect(s).

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Hort. Society Enterprise Ltd. RHS Garden; Wisley,Woking, Surrey GU236QB, UK.

Single gene converted (conversion). Single gene converted (conversion)plants refer to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a plant are recovered in additionto the single gene transferred into the plant via the backcrossingtechnique or via genetic engineering. A single gene converted plant canalso be referred to a plant obtained though mutagenesis or through theuse of some new breeding techniques, whereas the single gene convertedplant has essentially all of the desired morphological and physiologicalcharacteristics of the original variety in addition to the single geneor nucleotide sequence muted or engineered through the New BreedingTechniques.

Susceptible to disease(s) and or insect(s). A lettuce plant that issusceptible to disease(s) and or insect(s) is defined as a lettuce plantthat has the inability to restrict the growth and development ofspecific disease(s) and or insect(s). Plants that are susceptible willshow damage when infected and are more likely to have heavy damage undermoderate levels of specific disease(s) and or insect(s).

Tip burn. Means a browning of the edges or tips of lettuce leaves thatis a physiological response to a lack of calcium

Tolerance to abiotic stresses. A lettuce plant that is tolerant toabiotic stresses has the ability to endure abiotic stress withoutserious consequences for growth, appearance and yield.

Uniformity. Uniformity, as used herein, describes the similarity betweenplants or plant characteristics which can be a described by qualitativeor quantitative measurements.

Variety. A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties ofplants).

Lettuce Plants

Most cultivated forms of lettuce belong to the highly polymorphicspecies Lactuca sativa which is grown for its edible head and leaves. Asa crop, lettuce is grown commercially wherever environmental conditionspermit the production of an economically viable yield. Lettuce is theworld's most popular salad. In the United States, the principal growingregions are California and Arizona which produce approximately 329,000acres out of a total annual acreage of more than 333,000 acres (USDA,2005). Fresh lettuce is available in the United States year-roundalthough the greatest supply is from May through October. For plantingpurposes, the lettuce season is typically divided into three categories,early, mid and late, with the coastal areas planting from January toAugust, and the desert regions from August to December. Lettuce isconsumed nearly exclusively as fresh, raw product, and occasionally as acooked vegetable. Baby leaf or spring mix lettuce is an increasinglypopular crop as worldwide baby leaf lettuce consumption continues toincrease. Spring mix lettuce refers to lettuce that is grown in highconcentrations and harvested at a very young or ‘baby leaf’ stage,typically 30 to 45 days after planting. The plantings are often done onwider 80 to 84 inch beds and often contain up to one million plants peracre. Compared to iceberg or romaine plantings, where they are typicallyharvested 60 to 100 days after planting, with a population of roughly25,000 to 30,000 plants per acre. Spring mix plantings often includemultiple types of lettuces, all harvested when the leaves are young andtender. These plantings can include green romaine, red romaine, darklolla rossa, tango, green leaf, and red leaf types. Spring mix fieldsare most often harvested mechanically and the harvested leaves arepacked in plastic totes, where they are transported to a processingfacility where they are washed, processed and mixed according to thesalad recipe.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositaefamily). Lettuce is related to chicory, sunflower, aster, dandelion,artichoke, and chrysanthemum. Sativa is one of about 300 species in thegenus Lactuca. There are several morphological types of lettuce. TheCrisphead group includes the Iceberg and Batavian types. Iceberg lettucehas a large, firm head with a crisp texture and a white or creamy yellowinterior. Batavian lettuce predates Iceberg lettuce and has a smallerand less firm head. The Butterhead group has a small, soft head with analmost oily texture. Romaine lettuce, also known as Cos lettuce, haselongated upright leaves forming a loose, loaf-shaped head and the outerleaves are usually dark green. Leaf lettuce comes in many varieties,none of which form a head. There are three types of lettuce which areseldom seen in the United States: Latin lettuce, which looks like across between Romaine and Butterhead; Stem lettuce, which has long,narrow leaves and thick, edible stems; and Oilseed lettuce, which is aprimitive type of lettuce grown for its large seeds that are pressed toobtain oil.

Lactuca sativa is normally a simple diploid species with nine pairs ofchromosomes (2N=18). However, haploidy and polyploidy lettuce plants arealso part of the present invention. Lettuce is an obligateself-pollinating species which means that pollen is shed before stigmaemergence, assuring 100% self-fertilization. Since each lettuce floweris an aggregate of about 10-20 individual florets (typical of theCompositae family), manual removal of the anther tubes containing thepollen is tedious. As a result, a modified method of misting to wash offthe pollen prior to fertilization is needed to assure crossing orhybridization. Flowers to be used for crossings are selected about 60-90minutes after sunrise. Selection criteria include plants with openflowers, where the stigma has emerged and pollen is visibly attached toa single stigma (there are about 10-20 stigma). Pollen grains are washedoff using 3-4 pumps of water from a spray bottle and with enoughpressure to dislodge the pollen grains without damaging the style.Excess water is then dried off using clean paper towels and about 30minutes later, the styles spring back up and the two lobes of the stigmaare visibly open in a “V” shape. Pollen from another variety or donorparent is then introduced by gently rubbing the stigma and style of thedonor parent to the maternal parent. Most pertinent informationincluding dates and pedigree are then secured to the flowers using tags.

Hybrid vigor has been documented in lettuce and hybrids will be gainingmore and more popularity amongst farmers with uniformity of plantcharacteristics.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm.

In lettuce, these important traits may include increased head size andweight, higher seed yield, improved color, resistance to diseases andinsects, tolerance to drought and heat, better post-harvest shelf-lifeof the leaves, better standing ability in the field, better uniformity,and better agronomic quality.

In some embodiments, particularly desirable traits that may beincorporated by this invention are improved resistance to differentviral, fungal, and bacterial pathogens. Important diseases include butare not limited to fungi such as Bremia lactucae, Fusarium oxysporum,Sclerotinia minor or sclerotorum, Botrytis cinerea, Rhizictonia solani,Microdochium panattonianum, Verticiulium dahliae, Erysiphe chicocearumor Pithium tracheiphilum, virus, such as LMV (lettuce mosaic virus),TSWV (tomato potted wilt virus), “Big vein” (composed of LBW (lettucebig vein virus) and MILV (miratiori lettuce virus)), TBSV (tomato bushystunt virus), LNSV (lettuce necrotic stunt virus), TuMV (turnip mosaicvirus), CMV (cucumber mosaic virus) or BWYV (beet western yellowsvirus), bacteria such as Pseudomonas, Xanthomonas or Rhizomonas.Improved resistance to insect pests is another desirable trait that maybe incorporated into new lettuce plants developed by this invention.Insect pests affecting the various species of lettuce include Nasonoviaribisnigri, Myzus persicae, Macrosiphum euphorbia, Nematodespratylenchus or meloidogyne, leafminers: Liriomyza huidobrensis orPemphigus busarius.

Other desirable traits include traits related to improved lettuce plantsand parts thereof. A non-limiting list of lettuce phenotypes used duringbreeding selection includes:

-   -   Tomato Bushy Stunt (TBSV) resistance or tolerance. TBSV is a        viral disease which causes stunting of growth, leaf mottling,        and deformed or absent heads. When associated with Lettuce        Necrotic Stunt Virus (LNSV), another soil born virus, Tomato        Bushy Stunt leads to the disease known as Dieback (Simko et        al., 2010. HortScience 45(2): 670-672), resulting in mottling,        yellowing, and necrosis of older leaves, stunting of the plant,        and eventually death    -   Tip Burn tolerance. Tip burn tolerance is a tolerance to an        abiotic disorder caused by calcium deficiency in growing tissues        and resulting in the browning, up to black color, of the margins        of young, maturing leaves in head and leaf lettuces. The brown        area may be limited to a few small spots at or near the leaf        margin, or the entire edge of the leaf may be affected. The term        tip burn is usually used to refer to the browning in the        internal leaves of the plant. Tip burn is also caused by        environmental conditions that reduce transpiration such as foggy        conditions and soil water stress (source: UC Pest Management        Guidelines)    -   Fringe burn tolerance. Fringe burn tolerance is tolerance to        brown discoloration on the outer edge of the lettuce leaf.        Fringe burn may be limited to a few spots or cover the entire        edge of the leaf. The term Fringe burn is usually used to refer        to browning on the external leaves of the plant.

Lettuce Breeding

The goal of lettuce breeding is to develop new, unique and superiorlettuce cultivar and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. Another method used to developnew, unique and superior lettuce cultivar occurs when the breederselects and crosses two or more parental lines followed by haploidinduction and chromosome doubling that result in the development ofdihaploid cultivars. The breeder can theoretically generate billions ofdifferent genetic combinations via crossing, selfing and mutations andthe same is true for the utilization of the dihaploid breeding method.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarsdeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting cultivars he develops, exceptpossibly in a very gross and general fashion. This unpredictabilityresults in the expenditure of large research monies to develop superiornew lettuce cultivars.

The development of commercial lettuce cultivar requires the developmentand selection of lettuce plants, the crossing of these plants, and theevaluation of the crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes or through the dihaploidbreeding method followed by the selection of desired phenotypes. The newcultivars are evaluated to determine which have commercial potential.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

i Pedigree Selection

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents possessing favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). The dihaploid breedingmethod could also be used. Selection of the best individuals is usuallybegun in the F₂ population; then, beginning in the F₃, the bestindividuals in the best families are selected. Replicated testing offamilies, or hybrid combinations involving individuals of thesefamilies, often follows in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease of new cultivars. Similarly, the development of new cultivarsthrough the dihaploid system requires the selection of the cultivarsfollowed by two to five years of testing in replicated plots.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F2 to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F2 individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F2 plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or morefruit containing seed from each plant in a population and blend themtogether to form a bulk seed lot. Part of the bulked seed is used toplant the next generation and part is put in reserve. The procedure hasbeen referred to as modified single-seed descent or the bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster than removing one seed from each fruit by handfor the single seed procedure. The multiple-seed procedure also makes itpossible to plant the same number of seeds of a population eachgeneration of inbreeding. Enough seeds are harvested to make up forthose plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son, pp. 115-161; N. W. Simmonds, 1979, Principles of CropImprovement, Longman Group Limited; W. R. Fehr, 1987, Principles of CropDevelopment, Macmillan Publishing Co.; N. F. Jensen, 1988, PlantBreeding Methodology, John Wiley & Sons).

ii Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype recurrent parent andthe trait of interest from the donor parent are selected and repeatedlycrossed (backcrossed) to the recurrent parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent.

When the term lettuce cultivar is used in the context of the presentinvention, this also includes any lettuce cultivar plant where one ormore desired trait has been introduced through backcrossing methods,whether such trait is a naturally occurring one, a mutant, a transgenicone or a gene or a nucleotide sequence modified by the use of NewBreeding Techniques. Backcrossing methods can be used with the presentinvention to improve or introduce one or more characteristic into thelettuce cultivar of the present invention. The term “backcrossing” asused herein refers to the repeated crossing of a hybrid progeny back tothe recurrent parent, i.e., backcrossing one, two, three, four, five,six, seven, eight, nine, or more times to the recurrent parent. Theparental lettuce cultivar plant which contributes the gene or the genesfor the desired characteristic is termed the nonrecurrent or donorparent. This terminology refers to the fact that the nonrecurrent parentis used one time in the backcross protocol and therefore does not recur.The parental lettuce cultivar to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol.

In a typical backcross protocol, the original cultivar of interest(recurrent parent) is crossed to a second cultivar (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a lettuce plant isobtained wherein all the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, generally determined at a 5% significance level when grown in thesame environmental conditions, in addition to the gene or genestransferred from the nonrecurrent parent. It has to be noted that some,one, two, three or more, self-pollination and growing of populationmight be included between two successive backcrosses. Indeed, anappropriate selection in the population produced by theself-pollination, i.e. selection for the desired trait and physiologicaland morphological characteristics of the recurrent parent might beequivalent to one, two or even three additional backcrosses in acontinuous series without rigorous selection, saving then time, moneyand effort to the breeder. A non-limiting example of such a protocolwould be the following: a) the first generation F1 produced by the crossof the recurrent parent A by the donor parent B is backcrossed to parentA, b) selection is practiced for the plants having the desired trait ofparent B, c) selected plant are self-pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,five, six, seven, eight, nine, or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and the physiological and morphological characteristics of parent A.Step (c) may or may not be repeated and included between the backcrossesof step (d).

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred parental line in order to find it then in the hybridmade thereof. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass visual inspection, simplecrossing, but also follow up of the characteristic(s) throughgenetically associated markers and molecular assisted breeding tools.For example, selection of progeny containing the transferred trait isdone by direct selection, visual inspection for a trait associated witha dominant allele, while the selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic in corn, require selfing the progeny to determine whichplant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new lettuce cultivar plantaccording to the invention but that can be improved by backcrossingtechniques. Single gene traits may or may not be transgenic. Examples ofthese traits include but are not limited to, male sterility (such as aPR glucanase gene or the ms1, ms2, ms3, ms4, ms5, ms7 genes), herbicideresistance (such as bar or PAT genes). Gene controlling resistance tothe lettuce leaf aphid Nasonovia ribisnigri (Nr gene) can be found inVan der Arend and Schijndel in Breeding for Resistance to insects andMites, IOBC wprs Bulletin 22(10), 35-43 (1999). Other traits forresistance or tolerance to an infection by a virus, a bacterium, aninsect or a fungus, might be obtained from the genes for resistance toBremia Dm10, R17, Dm5, Dm8, R36, R37 (genes located on cluster 1 ofLactuca sativa), Dm1, Dm2, Dm3, Dm6, Dm14, Dm15, Dm16, Dm18 (geneslocated on cluster 2 of Lactuca sativa), Dm4, Dm7, Dm11, R38 (geneslocated on cluster 4 of Lactuca sativa); or the Tu gene for resistanceto TuMV located on cluster 1; or from the genes mol.1 and mol.2 forresistance to LMV located on cluster 4. Clusters 1, 2 and 4 cited abovehave been defined by Michelmore R. W. (Plant Pathol, 1987, vol. 36, no4: 499-514 [4], Theor. Appl. Genet., 1993, vol. 85, No 8: 985-993. Thesegenes are generally inherited through the nucleus. Some other singlegene traits are described in U.S. Pat. Nos. 5,777,196, 5,948,957, and5,969,212, the disclosures of which are specifically hereby incorporatedby reference.

In 1981, the backcross method of breeding counted for 17% of the totalbreeding effort for inbred line development in the United States,accordingly to, Hallauer, A. R. et al. (1988) “Corn Breeding” Corn andCorn Improvement, No. 18, pp. 463-481.

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R. W. Allard's 1960 book,published by John Wiley & Sons, Inc, Principles of Plant Breeding). Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a parental line of a hybrid variety with exactly oressentially the same adaptation, yielding ability and qualitycharacteristics of the recurrent parent but superior to that parent inthe particular characteristic(s) for which the improvement program wasundertaken. Therefore, this method provides the plant breeder with ahigh degree of genetic control of his work.

The method is scientifically exact because the morphological andagricultural features of the improved variety could be described inadvance and because a similar variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method”, 1930 Jour. Amer. Soc.Agron., 22: 289-244).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will betheoretically modified only with regards to genes being transferred,which are maintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from ‘Hope’ wheat to ‘Bart wheat’ and even pursuing thebackcrosses with the transfer of bunt resistance to create ‘Bart 38’,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in California Commonalfalfa to create ‘Caliverde’. This new ‘Caliverde’ variety producedthrough the backcross process is indistinguishable from CaliforniaCommon except for its resistance to the three named diseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred or when usingmolecular markers that can identify the trait of interest.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,color characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, ‘Calady’, has been produced by Jones andDavis. As dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e. grain size. ‘Lady Wright’, along grain variety was used as the donor parent and ‘Coloro’, a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety ‘Calady’ was produced.

iii Open-Pollinated Populations

The improvement of open-pollinated populations of such crops as rye,many maizes and sugar beets, herbage grasses, legumes such as alfalfaand clover, and tropical tree crops such as cacao, coconuts, oil palmand some rubber, depends essentially upon changing gene-frequenciestowards fixation of favorable alleles while maintaining a high (but farfrom maximal) degree of heterozygosity.

Uniformity in such populations is impossible and trueness-to-type in anopen-pollinated variety is a statistical feature of the population as awhole, not a characteristic of individual plants. Thus, theheterogeneity of open-pollinated populations contrasts with thehomogeneity (or virtually so) of inbred lines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes to flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement.

First, there is the situation in which a population is changed en masseby a chosen selection procedure. The outcome is an improved populationthat is indefinitely propagated by random-mating within itself inisolation.

Second, the synthetic variety attains the same end result as populationimprovement, but is not itself propagated as such; it has to bereconstructed from parental lines or clones. These plant breedingprocedures for improving open-pollinated populations are well known tothose skilled in the art and comprehensive reviews of breedingprocedures routinely used for improving cross-pollinated plants areprovided in numerous texts and articles, including: Allard, Principlesof Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds, Principlesof Crop Improvement, Longman Group Limited (1979); Hallauer and Miranda,Quantitative Genetics in Maize Breeding, Iowa State University Press(1981); and, Jensen, Plant Breeding Methodology, John Wiley & Sons, Inc.(1988).

A) Mass Selection

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued. In massselection, desirable individual plants are chosen, harvested, and theseed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

B) Synthetics

A synthetic variety is produced by intercrossing a number of genotypesselected for good combining ability in all possible hybrid combinations,with subsequent maintenance of the variety by open pollination. Whetherparents are (more or less inbred) seed-propagated lines, as in somesugar beet and beans (Vicia) or clones, as in herbage grasses, cloversand alfalfa, makes no difference in principle. Parents are selected ongeneral combining ability, sometimes by test crosses or toperosses, moregenerally by polycrosses. Parental seed lines may be deliberately inbred(e.g. by selfing or sib crossing). However, even if the parents are notdeliberately inbred, selection within lines during line maintenance willensure that some inbreeding occurs. Clonal parents will, of course,remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or more cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enters a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

iv. Hybrids

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugarbeet, sunflower andbroccoli. Hybrids can be formed in a number of different ways, includingby crossing two parents directly (single cross hybrids), by crossing asingle cross hybrid with another parent (three-way or triple crosshybrids), or by crossing two different hybrids (four-way or double crosshybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

Hybrid commercial lettuce seed is produced by insect pollination, seeU.S. Pat. No. 8,716,551 which is specifically hereby incorporated byreference.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F1progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F1 hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor and uniformity exhibited byF1 hybrids is lost in the next generation (F2). Consequently, seed fromF2 hybrid varieties is not used for planting stock.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

v. Bulk Segregation Analysis (BSA)

BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, isa method described by Michelmore et al. (Michelmore et al., 1991,Identification of markers linked to disease-resistance genes by bulkedsegregant analysis: a rapid method to detect markers in specific genomicregions by using segregating populations. Proceedings of the NationalAcademy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie etal., 1999, Journal of Experimental Botany, 50(337): 1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F2, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to virus), and theother from the individuals having reversed phenotype (e.g., susceptibleto virus), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs, SNPs or SSRs). Markers linkedto the phenotype can be identified and used for breeding or QTL mapping.

vi. Hand-Pollination Method

Hand pollination describes the crossing of plants via the deliberatefertilization of female ovules with pollen from a desired male parentplant. In some embodiments the donor or recipient female parent and thedonor or recipient male parent line are planted in the same field. Insome embodiments the donor or recipient female parent and the donor orrecipient male parent line are planted in the same greenhouse. Theinbred male parent can be planted earlier than the female parent toensure adequate pollen supply at the pollination time. In someembodiments, the male parent and female parent can be planted at a ratioof 1 male parent to 4-10 female parents. The male parent may be plantedat the top of the field for efficient male flower collection duringpollination. Pollination is started when the female parent flower isready to be fertilized. Female flower buds that are ready to open in thefollowing days are identified, covered with paper cups or small paperbags that prevent bee or any other insect from visiting the femaleflowers, and marked with any kind of material that can be easily seenthe next morning. In some embodiments, this process is best done in theafternoon. The male flowers of the male parent are collected in theearly morning before they are open and visited by pollinating insects.The covered female flowers of the female parent, which have opened, areun-covered and pollinated with the collected fresh male flowers of themale parent, starting as soon as the male flower sheds pollen. Thepollinated female flowers are again covered after pollination to preventbees and any other insects visit. The pollinated female flowers are alsomarked. The marked flowers are harvested. In some embodiments, the malepollen used for fertilization has been previously collected and stored.

vii. Bee-Pollination Method

Using the bee-pollination method, the parent plants are usually plantedwithin close proximity. In some embodiments more female plants areplanted to allow for a greater production of seed. Insects are placed inthe field or greenhouses for transfer of pollen from the male parent tothe female flowers of the female parent.

viii. Targeting Induced Local Lesions in Genomes (TILLING)

Breeding schemes of the present application can include crosses withTILLING® plant cultivars. TILLING® is a method in molecular biology thatallows directed identification of mutations in a specific gene. TILLING®was introduced in 2000, using the model plant Arabidopsis thaliana.TILLING® has since been used as a reverse genetics method in otherorganisms such as zebrafish, corn, wheat, rice, soybean, tomato andlettuce.

The method combines a standard and efficient technique of mutagenesiswith a chemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with asensitive DNA screening-technique that identifies single base mutations(also called point mutations) in a target gene. EcoTILLING is a methodthat uses TILLING® techniques to look for natural mutations inindividuals, usually for population genetics analysis (see Comai, etal., 2003 The Plant Journal 37, 778-786; Gilchrist et al. 2006 Mol.Ecol. 15, 1367-1378; Mejlhede et al. 2006 Plant Breeding 125, 461-467;Nieto et al. 2007 BMC Plant Biology 7, 34-42, each of which isincorporated by reference hereby for all purposes). DEcoTILLING is amodification of TILLING® and EcoTILLING which uses an inexpensive methodto identify fragments (Garvin et al., 2007, DEco-TILLING: An inexpensivemethod for SNP discovery that reduces ascertainment bias. MolecularEcology Notes 7, 735-746).

The TILLING® method relies on the formation of heteroduplexes that areformed when multiple alleles (which could be from a heterozygote or apool of multiple homozygotes and heterozygotes) are amplified in a PCR,heated, and then slowly cooled. As DNA bases are not pairing at themismatch of the two DNA strands (the induced mutation in TILLING® or thenatural mutation or SNP in EcoTILLING), they provoke a shape change inthe double strand DNA fragment which is then cleaved by single strandednucleases. The products are then separated by size on several differentplatforms.

Several TILLING® centers exists over the world that focus onagriculturally important species: UC Davis (USA), focusing on Rice;Purdue University (USA), focusing on Maize; University of BritishColumbia (CA), focusing on Brassica napus; John Innes Centre (UK),focusing on Brassica rapa; Fred Hutchinson Cancer Research, focusing onArabidopsis; Southern Illinois University (USA), focusing on Soybean;John Innes Centre (UK), focusing on Lotus and Medicago; and INRA(France), focusing on Pea and Tomato.

More detailed description on methods and compositions on TILLING® can befound in U.S. Pat. No. 5,994,075, US 2004/0053236 A1, WO 2005/055704,and WO 2005/048692, each of which is hereby incorporated by referencefor all purposes.

Thus in some embodiments, the breeding methods of the present disclosureinclude breeding with one or more TILLING plant lines with one or moreidentified mutations.

viii Mutation Breeding

Mutation breeding is another method of introducing new variation andsubsequent traits into lettuce plants. Mutations that occurspontaneously or are artificially induced can be useful sources ofvariability for a plant breeder. The goal of artificial mutagenesis isto increase the rate of mutation for a desired characteristic. Mutationrates can be increased by many different means or mutating agentsincluding temperature, long-term seed storage, tissue cultureconditions, radiation (such as X-rays, Gamma rays, neutrons, Betaradiation, or ultraviolet radiation), chemical mutagens (such as baseanalogs like 5-bromo-uracil), antibiotics, alkylating agents (such assulfur mustards, nitrogen mustards, epoxides, ethyleneamines, sulfates,sulfonates, sulfones, or lactones), azide, hydroxylamine, nitrous acidor acridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found in W. R.Fehr, 1993, Principles of Cultivar Development, Macmillan Publishing Co.

New breeding techniques such as the ones involving the uses of ZincFinger Nucleases or oligonucleotide directed mutagenesis shall also beused to generate genetic variability and introduce new traits intolettuce varieties.

ix. Double Haploids and Chromosome Doubling

One way to obtain homozygous plants without the need to cross twoparental lines followed by a long selection of the segregating progeny,and/or multiple backcrossing is to produce haploids and then double thechromosomes to form doubled haploids. Haploid plants can occurspontaneously, or may be artificially induced via chemical treatments orby crossing plants with inducer lines (Seymour et al. 2012, PNAS vol109, pg 4227-4232; Zhang et al., 2008 Plant Cell Rep. Dec 27(12)1851-60). The production of haploid progeny can occur via a variety ofmechanisms which can affect the distribution of chromosomes duringgamete formation. The chromosome complements of haploids sometimesdouble spontaneously to produce homozygous doubled haploids (DHs).Mixoploids, which are plants which contain cells having differentploidies, can sometimes arise and may represent plants that areundergoing chromosome doubling so as to spontaneously produce doubledhaploid tissues, organs, shoots, floral parts or plants. Another commontechnique is to induce the formation of double haploid plants with achromosome doubling treatment such as colchicine (El-Hennawy et al.,2011 Vol 56, issue 2 pg 63-72; Doubled Haploid Production in Crop Plants2003 edited by Maluszynski ISBN 1-4020-1544-5). The production ofdoubled haploid plants yields highly uniform cultivars and is especiallydesirable as an alternative to sexual inbreeding of longer-generationcrops. By producing doubled haploid progeny, the number of possible genecombinations for inherited traits is more manageable. Thus, an efficientdoubled haploid technology can significantly reduce the time and thecost of inbred and cultivar development.

x. Protoplast Fusion

In another method for breeding plants, protoplast fusion can also beused for the transfer of trait-conferring genomic material from a donorplant to a recipient plant. Protoplast fusion is an induced orspontaneous union, such as a somatic hybridization, between two or moreprotoplasts (cells of which the cell walls are removed by enzymatictreatment) to produce a single bi- or multi-nucleate cell. The fusedcell that may even be obtained with plant species that cannot beinterbred in nature is tissue cultured into a hybrid plant exhibitingthe desirable combination of traits.

xi. Embryo Rescue

Alternatively, embryo rescue may be employed in the transfer ofresistance-conferring genomic material from a donor plant to a recipientplant. Embryo rescue can be used as a procedure to isolate embryos fromcrosses to rapidly move to the next generation of backcrossing orselfing or wherein plants fail to produce viable seed. In this process,the fertilized ovary or immature seed of a plant is tissue cultured tocreate new plants (see Pierik, 1999, In Vitro Culture of Higher Plants,Springer, ISBN 079235267, 9780792352679, which is incorporated herein byreference in its entirety).

Breeding Evaluation

Each breeding program can include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for use as parents in new commercial cultivars; those stilldeficient in a few traits may be used as parents to produce newpopulations for further selection.

In one embodiment, the plants are selected on the basis of one or morephenotypic traits. Skilled persons will readily appreciate that suchtraits include any observable characteristic of the plant, including forexample growth rate, vigor, plant health, maturity, plant height, leafcoverage, weight, total yield, color, taste, sugar levels, aroma, smell,changes in the production of one or more compounds by the plant(including for example, metabolites, proteins, drugs, carbohydrates,oils, and any other compounds).

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

It should be appreciated that in certain embodiments, plants may beselected based on the absence, suppression or inhibition of a certainfeature or trait (such as an undesirable feature or trait) as opposed tothe presence of a certain feature or trait (such as a desirable featureor trait).

Selecting plants based on genotypic information is also envisaged (forexample, including the pattern of plant gene expression, genotype, orpresence of genetic markers). Where the presence of one or more geneticmarker is assessed, the one or more marker may already be known and/orassociated with a particular characteristic of a plant; for example, amarker or markers may be associated with an increased growth rate ormetabolite profile. This information could be used in combination withassessment based on other characteristics in a method of the disclosureto select for a combination of different plant characteristics that maybe desirable. Such techniques may be used to identify novel quantitativetrait loci (QTLs). By way of example, plants may be selected based ongrowth rate, size (including but not limited to weight, height, leafsize, stem size, branching pattern, or the size of any part of theplant), general health, survival, tolerance to adverse physicalenvironments and/or any other characteristic, as described hereinbefore.

Further non-limiting examples include selecting plants based on: speedof seed germination; quantity of biomass produced; increased root,and/or leaf/shoot growth that leads to an increased yield (herbage orgrain or fiber or oil) or biomass production; effects on plant growththat results in an increased seed yield for a crop; effects on plantgrowth which result in an increased head yield; effects on plant growththat lead to an increased resistance or tolerance to disease includingfungal, viral or bacterial diseases, to mycoplasma, or to pests such asinsects, mites or nematodes in which damage is measured by decreasedfoliar symptoms such as the incidence of bacterial or fungal lesions, orarea of damaged foliage or reduction in the numbers of nematode cysts orgalls on plant roots, or improvements in plant yield in the presence ofsuch plant pests and diseases; effects on plant growth that lead toincreased metabolite yields; effects on plant growth that lead toimproved aesthetic appeal which may be particularly important in plantsgrown for their form, color or taste, for example the color intensity oflettuce leaves, or the taste of said leaves.

Molecular Breeding Evaluation Techniques

Selection of plants based on phenotypic or genotypic information may beperformed using techniques such as, but not limited to: high through-putscreening of chemical components of plant origin, sequencing techniquesincluding high through-put sequencing of genetic material, differentialdisplay techniques (including DDRT-PCR, and DD-PCR), nucleic acidmicroarray techniques, RNA-seq (Transcriptome Sequencing), qRTPCR(quantitative real time PCR).

In one embodiment, the evaluating step of a plant breeding programinvolves the identification of desirable traits in progeny plants.Progeny plants can be grown in, or exposed to conditions designed toemphasize a particular trait (e.g. drought conditions for droughttolerance, lower temperatures for freezing tolerant traits). Progenyplants with the highest scores for a particular trait may be used forsubsequent breeding steps.

In some embodiments, plants selected from the evaluation step canexhibit a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120% or more improvement in aparticular plant trait compared to a control plant.

In other embodiments, the evaluating step of plant breeding comprisesone or more molecular biological tests for genes or other markers. Forexample, the molecular biological test can involve probe hybridizationand/or amplification of nucleic acid (e.g., measuring nucleic aciddensity by Northern or Southern hybridization, PCR) and/or immunologicaldetection (e.g., measuring protein density, such as precipitation andagglutination tests, ELISA (e.g., Lateral Flow test or DAS-ELISA),Western blot, immune labeling, immunosorbent electron microscopy (ISEM),and/or dot blot).

The procedure to perform a nucleic acid hybridization, an amplificationof nucleic acid (e.g., PCR, RT-PCR) or an immunological detection (e.g.,precipitation and agglutination tests, ELISA (e.g., Lateral Flow test orDAS-ELISA), Western blot, RIA, immunogold or immunofluorescent labeling,immunosorbent electron microscopy (ISEM), and/or dot blot tests) areperformed as described elsewhere herein and well-known by one skilled inthe art.

In one embodiment, the evaluating step comprises PCR (semi-quantitativeor quantitative), wherein primers are used to amplify one or morenucleic acid sequences of a desirable gene, or a nucleic acid associatedwith said gene, or QTL or a desirable trait (e.g., a co-segregatingnucleic acid, or other marker).

In another embodiment, the evaluating step comprises immunologicaldetection (e.g., precipitation and agglutination tests, ELISA (e.g.,Lateral Flow test or DAS-ELISA), Western blot, MA, immuno labeling(gold, fluorescent, or other detectable marker), immunosorbent electronmicroscopy (ISEM), and/or dot blot), wherein one or more gene ormarker-specific antibodies are used to detect one or more desirableproteins. In one embodiment, said specific antibody is selected from thegroup consisting of polyclonal antibodies, monoclonal antibodies,antibody fragments, and combination thereof.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be utilizedin the present disclosure to determine expression of a gene to assistduring the selection step of a breeding scheme. It is a variant ofpolymerase chain reaction (PCR), a laboratory technique commonly used inmolecular biology to generate many copies of a DNA sequence, a processtermed “amplification”. In RT-PCR, however, RNA strand is first reversetranscribed into its DNA complement (complementary DNA, or cDNA) usingthe enzyme reverse transcriptase, and the resulting cDNA is amplifiedusing traditional or real-time PCR.

RT-PCR utilizes a pair of primers, which are complementary to a definedsequence on each of the two strands of the mRNA. These primers are thenextended by a DNA polymerase and a copy of the strand is made after eachcycle, leading to logarithmic amplification.

RT-PCR includes three major steps. The first step is the reversetranscription (RT) where RNA is reverse transcribed to cDNA using areverse transcriptase and primers. This step is very important in orderto allow the performance of PCR since DNA polymerase can act only on DNAtemplates. The RT step can be performed either in the same tube with PCR(one-step PCR) or in a separate one (two-step PCR) using a temperaturebetween 40° C. and 50° C., depending on the properties of the reversetranscriptase used.

The next step involves the denaturation of the dsDNA at 95° C., so thatthe two strands separate and the primers can bind again at lowertemperatures and begin a new chain reaction. Then, the temperature isdecreased until it reaches the annealing temperature which can varydepending on the set of primers used, their concentration, the probe andits concentration (if used), and the cation concentration. The mainconsideration, of course, when choosing the optimal annealingtemperature is the melting temperature (Tm) of the primers and probes(if used). The annealing temperature chosen for a PCR depends directlyon length and composition of the primers. This is the result of thedifference of hydrogen bonds between A-T (2 bonds) and G-C(3 bonds). Anannealing temperature about 5 degrees below the lowest Tm of the pair ofprimers is usually used.

The final step of PCR amplification is the DNA extension from theprimers which is done by the thermostable Taq DNA polymerase usually at72° C., which is the optimal temperature for the polymerase to work. Thelength of the incubation at each temperature, the temperaturealterations and the number of cycles are controlled by a programmablethermal cycler. The analysis of the PCR products depends on the type ofPCR applied. If a conventional PCR is used, the PCR product is detectedusing for example agarose gel electrophoresis or other polymer gel likepolyacrylamide gels and ethidium bromide (or other nucleic acidstaining).

Conventional RT-PCR is a time-consuming technique with importantlimitations when compared to real time PCR techniques. This, combinedwith the fact that ethidium bromide has low sensitivity, yields resultsthat are not always reliable. Moreover, there is an increasedcross-contamination risk of the samples since detection of the PCRproduct requires the post-amplification processing of the samples.Furthermore, the specificity of the assay is mainly determined by theprimers, which can give false-positive results. However, the mostimportant issue concerning conventional RT-PCR is the fact that it is asemi or even a low quantitative technique, where the amplicon can bevisualized only after the amplification ends.

Real time RT-PCR provides a method where the amplicons can be visualizedas the amplification progresses using a fluorescent reporter molecule.There are three major kinds of fluorescent reporters used in real timeRT-PCR, general non specific DNA Binding Dyes such as SYBR Green I,TaqMan Probes and Molecular Beacons (including Scorpions).

The real time PCR thermal cycler has a fluorescence detection threshold,below which it cannot discriminate the difference between amplificationgenerated signal and background noise. On the other hand, thefluorescence increases as the amplification progresses and theinstrument performs data acquisition during the annealing step of eachcycle. The number of amplicons will reach the detection baseline after aspecific cycle, which depends on the initial concentration of the targetDNA sequence. The cycle at which the instrument can discriminate theamplification generated fluorescence from the background noise is calledthe threshold cycle (Ct). The higher is the initial DNA concentration,the lower its Ct will be.

Other forms of nucleic acid detection can include next generationsequencing methods such as DNA SEQ or RNA SEQ using any known sequencingplatform including, but not limited to: Roche 454, Solexa GenomeAnalyzer, AB SOLiD, Illumina GA/HiSeq, Ion PGM, Mi Seq, among others(Liu et al., 2012 Journal of Biomedicine and Biotechnology Volume 2012ID 251364; Franca et al., 2002 Quarterly Reviews of Biophysics 35 pg169-200; Mardis 2008 Genomics and Human Genetics vol 9 pg 387-402).

In other embodiments, nucleic acids may be detected with other highthroughput hybridization technologies including microarrays, gene chips,LNA probes, nanoStrings, and fluorescence polarization detection amongothers.

In some embodiments, detection of markers can be achieved at an earlystage of plant growth by harvesting a small tissue sample (e.g., branch,or leaf disk). This approach is preferable when working with largepopulations as it allows breeders to weed out undesirable progeny at anearly stage and conserve growth space and resources for progeny whichshow more promise. In some embodiments the detection of markers isautomated, such that the detection and storage of marker data is handledby a machine. Recent advances in robotics have also led to full serviceanalysis tools capable of handling nucleic acid/protein markerextractions, detection, storage and analysis.

Quantitative Trait Loci

Breeding schemes of the present application can include crosses betweendonor and recipient plants. In some embodiments said donor plantscontain a gene or genes of interest which may confer the plant with adesirable phenotype. The recipient line can be an elite line or cultivarhaving certain favorable traits for commercial production. In oneembodiment, the elite line may contain other genes that also impart saidline with the desired phenotype. When crossed together, the donor andrecipient plant may create a progeny plant with combined desirable lociwhich may provide quantitatively additive effect of a particularcharacteristic. In that case, QTL mapping can be involved to facilitatethe breeding process.

A QTL (quantitative trait locus) mapping can be applied to determine theparts of the donor plant's genome conferring the desirable phenotype,and facilitate the breeding methods. Inheritance of quantitative traitsor polygenic inheritance refers to the inheritance of a phenotypiccharacteristic that varies in degree and can be attributed to theinteractions between two or more genes and their environment. Though notnecessarily genes themselves, quantitative trait loci (QTLs) arestretches of DNA that are closely linked to the genes that underlie thetrait in question. QTLs can be molecularly identified to help mapregions of the genome that contain genes involved in specifying aquantitative trait. This can be an early step in identifying andsequencing these genes.

Typically, QTLs underlie continuous traits (those traits that varycontinuously, e.g. yield, height, level of resistance to virus, etc.) asopposed to discrete traits (traits that have two or several charactervalues, e.g. smooth vs. wrinkled peas used by Mendel in hisexperiments). Moreover, a single phenotypic trait is usually determinedby many genes. Consequently, many QTLs are associated with a singletrait.

A quantitative trait locus (QTL) is a region of DNA that is associatedwith a particular phenotypic trait Knowing the number of QTLs thatexplains variation in the phenotypic trait tells about the geneticarchitecture of a trait. It may tell that a trait is controlled by manygenes of small effect, or by a few genes of large effect or by a severalgenes of small effect and few genes of larger effect.

Another use of QTLs is to identify candidate genes underlying a trait.Once a region of DNA is identified as contributing to a phenotype, itcan be sequenced. The DNA sequence of any genes in this region can thenbe compared to a database of DNA for genes whose function is alreadyknown.

In a recent development, classical QTL analyses are combined with geneexpression profiling i.e. by DNA microarrays. Such expression QTLs(e-QTLs) describes cis- and trans-controlling elements for theexpression of often disease-associated genes. Observed epistatic effectshave been found beneficial to identify the gene responsible by across-validation of genes within the interacting loci with metabolicpathway- and scientific literature databases.

QTL mapping is the statistical study of the alleles that occur in alocus and the phenotypes (physical forms or traits) that they produce(see, Meksem and Kahl, The handbook of plant genome mapping: genetic andphysical mapping, 2005, Wiley-VCH, ISBN 3527311165, 9783527311163).Because most traits of interest are governed by more than one gene,defining and studying the entire locus of genes related to a trait giveshope of understanding what effect the genotype of an individual mighthave in the real world.

Statistical analysis is required to demonstrate that different genesinteract with one another and to determine whether they produce asignificant effect on the phenotype. QTLs identify a particular regionof the genome as containing one or several genes, i.e. a cluster ofgenes that is associated with the trait being assayed or measured. Theyare shown as intervals across a chromosome, where the probability ofassociation is plotted for each marker used in the mapping experiment.

To begin, a set of genetic markers must be developed for the species inquestion. A marker is an identifiable region of variable DNA. Biologistsare interested in understanding the genetic basis of phenotypes(physical traits). The aim is to find a marker that is significantlymore likely to co-occur with the trait than expected by chance, that is,a marker that has a statistical association with the trait. Ideally,they would be able to find the specific gene or genes in question, butthis is a long and difficult undertaking. Instead, they can more readilyfind regions of DNA that are very close to the genes in question. When aQTL is found, it is often not the actual gene underlying the phenotypictrait, but rather a region of DNA that is closely linked with the gene.

For organisms whose genomes are known, one might now try to excludegenes in the identified region whose function is known with somecertainty not to be connected with the trait in question. If the genomeis not available, it may be an option to sequence the identified regionand determine the putative functions of genes by their similarity togenes with known function, usually in other genomes. This can be doneusing BLAST, an online tool that allows users to enter a primarysequence and search for similar sequences within the BLAST database ofgenes from various organisms.

Another interest of statistical geneticists using QTL mapping is todetermine the complexity of the genetic architecture underlying aphenotypic trait. For example, they may be interested in knowing whethera phenotype is shaped by many independent loci, or by a few loci, andhow do those loci interact. This can provide information on how thephenotype may be evolving.

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization is possible due to DNA-DNAhybridization techniques (RFLP) and/or due to techniques using thepolymerase chain reaction (e.g. STS, SNPs, microsatellites, AFLP). Alldifferences between two parental genotypes will segregate in a mappingpopulation based on the cross of these parental genotypes. Thesegregation of the different markers may be compared and recombinationfrequencies can be calculated. The recombination frequencies ofmolecular markers on different chromosomes are generally 50%. Betweenmolecular markers located on the same chromosome the recombinationfrequency depends on the distance between the markers. A lowrecombination frequency usually corresponds to a low distance betweenmarkers on a chromosome. Comparing all recombination frequencies willresult in the most logical order of the molecular markers on thechromosomes. This most logical order can be depicted in a linkage map(Paterson, 1996, Genome Mapping in Plants. R. G. Landes, Austin.). Agroup of adjacent or contiguous markers on the linkage map that isassociated to a reduced disease incidence and/or a reduced lesion growthrate pinpoints the position of a QTL.

The nucleic acid sequence of a QTL may be determined by methods known tothe skilled person. For instance, a nucleic acid sequence comprisingsaid QTL or a resistance-conferring part thereof may be isolated from adonor plant by fragmenting the genome of said plant and selecting thosefragments harboring one or more markers indicative of said QTL.Subsequently, or alternatively, the marker sequences (or parts thereof)indicative of said QTL may be used as (PCR) amplification primers, inorder to amplify a nucleic acid sequence comprising said QTL from agenomic nucleic acid sample or a genome fragment obtained from saidplant. The amplified sequence may then be purified in order to obtainthe isolated QTL. The nucleotide sequence of the QTL, and/or of anyadditional markers comprised therein, may then be obtained by standardsequencing methods.

One or more such QTLs associated with a desirable trait in a donor plantcan be transferred to a recipient plant to incorporate the desirabletrait into progeny plants by transferring and/or breeding methods.

In one embodiment, an advanced backcross QTL analysis (AB-QTL) is usedto discover the nucleotide sequence or the QTLs responsible for theresistance of a plant. Such method was proposed by Tanksley and Nelsonin 1996 (Tanksley and Nelson, 1996, Advanced backcross QTL analysis: amethod for simultaneous discovery and transfer of valuable QTL fromun-adapted germplasm into elite breeding lines. Theor Appl Genet92:191-203) as a new breeding method that integrates the process of QTLdiscovery with variety development, by simultaneously identifying andtransferring useful QTL alleles from un-adapted (e.g., land races, wildspecies) to elite germplasm, thus broadening the genetic diversityavailable for breeding. AB-QTL strategy was initially developed andtested in tomato, and has been adapted for use in other crops includingrice, maize, wheat, pepper, barley, and bean. Once favorable QTL allelesare detected, only a few additional marker-assisted generations arerequired to generate near isogenic lines (NILs) or introgression lines(ILs) that can be field tested in order to confirm the QTL effect andsubsequently used for variety development.

Isogenic lines in which favorable QTL alleles have been fixed can begenerated by systematic backcrossing and introgressing of marker-defineddonor segments in the recurrent parent background. These isogenic linesare referred to as near isogenic lines (NILs), introgression lines(ILs), backcross inbred lines (BILs), backcross recombinant inbred lines(BCRIL), recombinant chromosome substitution lines (RCSLs), chromosomesegment substitution lines (CSSLs), and stepped aligned inbredrecombinant strains (STAIRSs). An introgression line in plant molecularbiology is a line of a crop species that contains genetic materialderived from a similar species. ILs represent NILs with relatively largeaverage introgression length, while BILs and BCRILs are backcrosspopulations generally containing multiple donor introgressions per line.As used herein, the term “introgression lines or ILs” refers to plantlines containing a single marker defined homozygous donor segment, andthe term “pre-ILs” refers to lines which still contain multiplehomozygous and/or heterozygous donor segments.

To enhance the rate of progress of introgression breeding, a geneticinfrastructure of exotic libraries can be developed. Such an exoticlibrary comprises a set of introgression lines, each of which has asingle, possibly homozygous, marker-defined chromosomal segment thatoriginates from a donor exotic parent, in an otherwise homogenous elitegenetic background, so that the entire donor genome would be representedin a set of introgression lines. A collection of such introgressionlines is referred as libraries of introgression lines or IL libraries(ILLs). The lines of an ILL cover usually the complete genome of thedonor, or the part of interest. Introgression lines allow the study ofquantitative trait loci, but also the creation of new varieties byintroducing exotic traits. High resolution mapping of QTL using ILLsenable breeders to assess whether the effect on the phenotype is due toa single QTL or to several tightly linked QTL affecting the same trait.In addition, sub-ILs can be developed to discover molecular markerswhich are more tightly linked to the QTL of interest, which can be usedfor marker-assisted breeding (MAB). Multiple introgression lines can bedeveloped when the introgression of a single QTL is not sufficient toresult in a substantial improvement in agriculturally important traits(Gur and Zamir, Unused natural variation can lift yield barriers inplant breeding, 2004, PLoS Biol.; 2(10):e245).

Tissue Culture

As it is well known in the art, tissue culture of lettuce can be usedfor the in vitro regeneration of lettuce plants. Tissues cultures ofvarious tissues of lettuce and regeneration of plants therefrom are wellknown and published. For example, reference may be had to Teng et al.,HortScience, 27: 9, 1030-1032 (1992), Teng et al., HortScience. 28: 6,669-671 (1993), Zhang et al., Journal of Genetics and Breeding, 46: 3,287-290 (1992), Webb et al., Plant Cell Tissue and Organ Culture, 38: 1,77-79 (1994), Curtis et al., Journal of Experimental Botany, 45: 279,1441-1449 (1994), Nagata et al., Journal for the American Society forHorticultural Science, 125: 6, 669-672 (2000). It is clear from theliterature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce lettuce plants having thephysiological and morphological characteristics of lettuce cultivarCABALLERO.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, leaves, stems,roots, root tips, anthers, pistils, meristematic cells, axillary buds,ovaries, seed coat, endosperm, hypocotyls, cotyledons and the like.Means for preparing and maintaining plant tissue culture are well knownin the art. By way of example, a tissue culture comprising organs hasbeen used to produce regenerated plants. U.S. Pat. Nos. 5,959,185,5,973,234, and 5,977,445 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

EXAMPLES

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

Example 1—Development of Lettuce Cultivar CABALLERO

Lettuce cultivar CABALLERO is a dark green iceberg, with large framewhich give excellent weight. It has nice round shape with good wrappingand good head protection. It is very uniform and consistent production.Thanks to its vigorous growing, it is suitable for production in coolweather condition such as winter season in the Southwest Deserts in theUSA.

Breeding History:

Lettuce cultivar CABALLERO has superior characteristics and wasdeveloped from the cross 68/23418 which correspond to across between24/187×8/20995. It was made in the summer in a greenhouse at VilmorinMikado Research facilities in La Ménitré, France. The F1 plants weregrown in greenhouse during the winter in La Ménitré. The F2 68/23418/01was sown in open field in spring of the second year of development inMade, Netherlands, with sowing number 12/16213. We have chosen 10 plantsidentified under code 16/16213/14 to 16/16213/23, grown in greenhouse inLa Ménitré during the summer. The F3 genotype 516/16213/16 was tested infall for Bremia resistance at Vilmorin pathological lab in La Ménitré,France. It was resistant to Bremia. Additionally, we provided DNAmarkers analysis to identify the Nr gene, that gives the resistance toaphid Nasonovia. This genotype was sown in open field in spring of thethird year in Made, Netherlands, under sowing number 13/16171. Sevenplants were selected in the plot, 13/16171/01 to/07. These plants weregrown under glasshouse at Vilmorin Research facilities in La Ménitré,France during summer. The F4 genotype 13/16171/03 was tested in fall forBremia resistance at Vilmorin pathological lab in La Ménitré, France. Itwas resistant to Bremia.

We have produced a F5 seed lot in large quantity in Vivy, France insummer of the fourth year. This F5 seed lot was tested on several Bremiaraces in November in La Ménitré. It was resistant to all of these Bremiaraces. This F5 seed lot, coded 13/16171/30, is corresponding to theIceberg lettuce variety CABALLERO.

Some of the agronomical criteria used to select the lettuce cultivarCABALLERO in various generations include the leaf green color andtexture, the vigor of the plant, the shape of the head, the weight ofthe head, the core length, the core axis, the balance of outer leave andhead size, the quality of the bottom, the uniformity.

The lettuce cultivar CABALLERO has shown uniformity and stability forthe traits, within the limits of environmental influence for the traitsas described in the following Variety Descriptive Information. Novariant traits have been observed or are expected for agronomicalimportant traits in lettuce cultivar CABALLERO.

Lettuce cultivar CABALLERO has the following morphologic and othercharacteristics, (based primarily on data collected in California, allexperiments done under the direct supervision of the applicant).

TABLE 1 Variety Description Information Plant: CABALLERO Type: IcebergSeed: Color: Black Cotyledon to Fourth Leaf Stage: Shape of cotyledon:Broad Shape of fourth leaf: Elongated Apical Margin: Entire BasalMargin: Entire Undulation: Slight Color: Dark green AnthocyaninDistribution: Absent Anthocyanin Concentration: N/A Rolling: PresentCupping: Uncupped Reflexing: None Harvest-Mature Out Leaf, Head, Core:Margin Incision Depth: Moderate Margin Indentation: Shallowly DentateUndulation of the Apical Margin: Moderate Color: Dark green AnthocyaninDistribution: Absent Anthocyanin Concentration: N/A Glossiness: ModerateBlistering: Moderate Leaf Thickness: Thick Trichomes: Absent (Smooth)Head Shape: Round Head Size Glass: Large Head per Carton: 24 HeadFirmness: Firm Butt Midrib: Flattened Maturity (No. of Days of FirstWater date to 95-105 Harvest, in Winter, Southwest deserts): Outer LeafLength (cm): 29.9 Out Leaf Width (cm): 28.5 Leaf Index: 1.06 Leaf Area:857.0 Head Weight (g): 710.4 Head height (cm): 13.35 Core Length (mm):3.13 Core Length (mm): 2.98 Seed Production Stage Seed stalk height (cm)43.7 Seed stalk spread (cm) 19.9 Adaptation: Yuma/Arizona Season: Winterseason in Yuma Soil type: adapted to most soil types Diseases: Tomatobushy stunt virus High Resistance Downy Mildew: Bl16-36 UE, 5-8 USNasonovia ribisnigri: Resistant to Nr0 Physiological/Stress: Bolting :Late bolting Tipburn: Tolerant

Example 2—Field Trials Characteristics of Lettuce Cultivar CABALLERO

Several traits and characteristics of lettuce cultivar CABALLERO werecompared to. The data was collected from various field locations in theUnited States. The field tests are experimental trials and have beenmade under supervision of the applicant. Compared to Serengeti andDesert Classic varieties, CABALLERO shows different head weight, frameleaf length at harvest maturity as well as in leaf area at harvestmaturity.

TABLE 2 Head Weight (g) at Harvest Maturity Trial NO. Trial 1 Trial 2Trial 3 Trial 4 Caballero (ICE 16171) 496 747 649 715 579 718 920 733694 840 897 721 667 805 650 647 654 694 562 660 594 822 583 714 709 793578 860 675 595 607 840 739 925 856 557 575 652 824 870 Serengeti 728681 907 990 550 720 781 625 552 952 622 652 581 819 547 840 421 726 755625 559 710 615 784 416 866 590 794 394 728 596 756 554 622 1148 826 577791 636 693 Desert Classic 485 402 649 812 689 629 689 657 510 695 549714 565 520 541 530 400 483 482 628 403 776 618 885 506 759 466 781 559706 634 550 430 606 863 656 640 683 333 412 Anova: Two-Factor WithReplication SUMMARY Trial 1 Trial 2 Trial 3 Trial 4 Total Caballero (ICE16171) Count 10.00 10.00 10.00 10.00 40.00 Sum 6382.00 7591.00 7126.007317.00 28416.00 Average 638.20 759.10 712.60 731.70 710.40 Variance5599.29 9528.10 20744.49 10095.57 12670.19 Serengeti Count 10.00 10.0010.00 10.00 40.00 Sum 5332.00 7615.00 7197.00 7585.00 27729.00 Average533.20 761.50 719.70 758.50 693.23 Variance 9953.96 9342.72 34776.4613109.39 24537.31 Desert Classic Count 10.00 10.00 10.00 10.00 40.00 Sum5187.00 6259.00 5824.00 6625.00 23895.00 Average 518.70 625.90 582.40662.50 597.38 Variance 9355.57 15207.66 20791.60 20308.50 18093.83 TotalCount 30.00 30.00 30.00 30.00 Sum 16901.00 21465.00 20147.00 21527.00Average 563.37 715.50 671.57 717.57 Variance 10663.14 14729.57 27804.3215196.46 ANOVA Source of Variation SS df MS F P-value F crit Variety296758.05 2.00 148379.03 9.96 0.00 3.08 Location 470098.80 3.00156699.60 10.52 0.00 2.69 Interaction 77333.55 6.00 12888.93 0.86 0.522.18 Within 1609319.60 108.00 14901.11 Total 2453510.00 119.00 Anovashows significant differences in head weight between varieties. Theaverage plant weight (g)of Caballero (ICE 16171), Serengeti and DesertClassic are 710.40, 693.23 and 597.38, respectively.

TABLE 3 Head Height (cm) at Harvest Maturity Trial NO. Trial 1 Trial 2Trial 3 Trial 4 Caballero (ICE 16171) 11 13 14 15 13 13 14 15 13 13 1413 13 13 12 13 14 12 13 13 14 14 13 14 13 14 14 13 12 14 13 13 12 15 1413 13 14 15 13 Serengeti 12 13 14 14 11 16 13 13 11 13 16 14 13 14 13 1511 14 15 14 12 15 13 15 11 14 13 13 12 15 14 13 12 14 13 14 13 15 14 14Desert Classic 13 13 14 15 12 14 13 14 13 15 13 14 13 14 12 15 13 14 1216 12 12 13 13 12 13 15 14 12 15 15 14 13 15 12 13 14 14 12 14 Anova:Two-Factor With Replication SUMMARY Trial 1 Trial 2 Trial 3 Trial 4Total Caballero (ICE 16171) Count 10 10 10 10 40 Sum 128 135 136 135 534Average 12.80 13.50 13.60 13.50 13.35 Variance 0.84 0.72 0.71 0.72 0.80Serengeti Count 10 10 10 10 40 Sum 118 143 138 139 538 Average 11.8014.30 13.80 13.90 13.45 Variance 0.62 0.90 1.07 0.54 1.69 Desert ClassicCount 10 10 10 10 40 Sum 127 139 131 142 539 Average 12.70 13.90 13.1014.20 13.48 Variance 0.46 0.99 1.43 0.84 1.23 Total Count 30 30 30 30Sum 373 417 405 416 Average 12.43 13.90 13.50 13.87 Variance 0.81 0.921.09 0.74 ANOVA Source of Variation SS df MS F P-value F crit Variety0.35 2 0.1750 0.2131 0.8084 3.0804 Location 42.29 3 14.0972 17.16460.0000 2.6887 Interaction 13.98 6 2.3306 2.8377 0.0132 2.1837 Within88.70 108 0.8213 Total 145.33 119 Anova shows no significant differencesin head height between Varieties. The average plant height (cm) ofCaballero (ICE 16171), Serengeti and Desert Classic are 13.35, 13.45 and13.48, respectively.

TABLE 4 Frame Leaf Length (cm) at Harvest Maturity Trial NO. Trial 1Trial 2 Trial 3 Trial 4 Caballero (ICE 16171) 27 26 35 30 28 30 30 35 2934 30 30 26 35 33 28 30 26 33 31 30 28 27 32 27 27 28 32 29 34 27 33 3030 29 29 27 35 33 26 Serengeti 29 30 36 31 26 33 29 30 28 35 30 35 30 3329 32 28 33 29 36 32 33 32 27 29 36 28 30 26 30 32 28 30 30 28 33 33 3432 35 Desert Classic 30 34 33 30 30 29 34 30 31 35 28 27 29 34 29 33 2732 29 33 30 32 28 33 31 36 29 31 29 32 26 33 31 35 32 31 31 35 31 37Anova: Two-Factor With Replication SUMMARY Trial 1 Trial 2 Trial 3 Trial4 Total Caballero (ICE 16171) Count 10.00 10.00 10.00 10.00 40.00 Sum283.00 305.00 305.00 306.00 1199.00 Average 28.30 30.50 30.50 30.6029.98 Variance 2.23 13.83 8.06 6.71 8.08 Serengeti Count 10.00 10.0010.00 10.00 40.00 Sum 291.00 327.00 305.00 317.00 1240.00 Average 29.1032.70 30.50 31.70 31.00 Variance 5.21 4.46 6.28 9.34 7.69 Desert ClassicCount 10.00 10.00 10.00 10.00 40.00 Sum 299.00 334.00 299.00 318.001250.00 Average 29.90 33.40 29.90 31.80 31.25 Variance 1.66 4.49 6.327.07 6.71 Total Count 30.00 30.00 30.00 30.00 Sum 873.00 966.00 909.00941.00 Average 29.10 32.20 30.30 31.37 Variance 3.27 8.65 6.49 7.48ANOVA Source of Variation SS df MS F P-value F crit Variety 36.52 2.0018.26 2.90 0.06 3.08 Location 162.23 3.00 54.08 8.58 0.00 2.69Interaction 33.35 6.00 5.56 0.88 0.51 2.18 Within 680.90 108.00 6.30Total 912.99 119.00 Anova shows significant differences in frame leaflength at harvest maturity stage between Varieties. The average maturedleaf length (cm) of Caballero, Serengeti and Desert Classic are 29.98,31.00 and 31.25, respectively.

TABLE 5 Frame Leaf Width (cm) at Harvest Maturity Trial NO. Trial 1Trial 2 Trial 3 Trial 4 Caballero (ICE 16171) 29 26 31 31 24 28 28 26 2935 27 29 30 34 27 30 29 28 29 27 27 29 30 37 28 27 26 23 25 30 30 28 2828 26 26 26 35 29 24 Serengeti 30 34 33 31 25 32 29 29 28 33 27 32 26 3131 31 29 29 28 33 28 32 30 26 25 29 30 29 24 31 27 28 26 29 28 35 27 3230 28 Desert Classic 35 28 28 26 23 30 30 33 28 32 30 27 30 31 30 32 2933 29 25 28 33 31 28 28 36 30 30 30 37 33 33 29 37 34 37 30 32 29 34Anova: Two-Factor With Replication SUMMARY Trial 1 Trial 2 Trial 3 Trial4 Total Caballero (ICE 16171) Count 10 10 10 10 40 Sum 275 300 283 2811139 Average 27.50 30.00 28.30 28.10 28.48 Variance 3.83 11.56 3.1216.10 8.87 Serengeti Count 10 10 10 10 40 Sum 268 312 293 302 1175Average 26.80 31.20 29.30 30.20 29.38 Variance 3.73 3.07 3.57 7.29 6.80Desert Classic Count 10 10 10 10 40 Sum 290 329 304 305 1228 Average29.00 32.90 30.40 30.50 30.70 Variance 8.67 8.99 3.38 15.39 10.42 TotalCount 30 30 30 30 Sum 833 941 880 888 Average 27.77 31.37 29.33 29.60Variance 5.91 8.79 3.89 13.21 ANOVA Source of Variation SS df MS FP-value F crit Variety 100.22 2 50.1083 6.7799 0.0017 3.0804 Location195.77 3 65.2556 8.8294 0.0000 2.6887 Interaction 23.78 6 3.9639 0.53630.7796 2.1837 Within 798.20 108 7.3907 Total 1117.97 119 Anova showssignificant differences in frame leaf width at harvest maturity stagebetween Varieties. The average matured leaf width (cm) of Caballero (ICE16171), Serengeti and Desert Classic are 28.48, 29.38 and 30.70,respectively.

TABLE 6 Frame Leaf Index calculated by dividing the leaf length by theleaf width Trial NO. Trial1 Trial2 Trial3 Trial4 Caballero (ICE 16171)0.93 1.00 1.13 0.97 1.17 1.07 1.07 1.35 1.00 0.97 1.11 1.03 0.87 1.031.22 0.93 1.03 0.93 1.14 1.15 1.11 0.97 0.90 0.86 0.96 1.00 1.08 1.391.16 1.13 0.90 1.18 1.07 1.07 1.12 1.12 1.04 1.00 1.14 1.08 Serengeti0.97 0.88 1.09 1.00 1.04 1.03 1.00 1.03 1.00 1.06 1.11 1.09 1.15 1.060.94 1.03 0.97 1.14 1.04 1.09 1.14 1.03 1.07 1.04 1.16 1.24 0.93 1.031.08 0.97 1.19 1.00 1.15 1.03 1.00 0.94 1.22 1.06 1.07 1.25 DesertClassic 0.86 1.21 1.18 1.15 1.30 0.97 1.13 0.91 1.11 1.09 0.93 1.00 0.971.10 0.97 1.03 0.93 0.97 1.00 1.32 1.07 0.97 0.90 1.18 1.11 1.00 0.971.03 0.97 0.86 0.79 1.00 1.07 0.95 0.94 0.84 1.03 1.09 1.07 1.09 Anova:Two-Factor With Replication SUMMARY Trial1 Trial2 Trial3 Trial4 TotalCaballero (ICE 16171) Count 10 10 10 10 40 Sum 10.34 10.17 10.80 11.0642.38 Average 1.03 1.02 1.08 1.11 1.06 Variance 0.01 0.00 0.01 0.03 0.01Serengeti Count 10 10 10 10 40 Sum 10.89 10.51 10.43 10.52 42.34 Average1.09 1.05 1.04 1.05 1.06 Variance 0.01 0.01 0.01 0.01 0.01 DesertClassic Count 10 10 10 10 40 Sum 10.41 10.22 9.88 10.55 41.06 Average1.04 1.02 0.99 1.06 1.03 Variance 0.02 0.01 0.01 0.02 0.01 Total Count30 30 30 30 Sum 31.65 30.90 31.11 32.13 Average 1.05 1.03 1.04 1.07Variance 0.01 0.01 0.01 0.02 ANOVA Source of Variation SS df MS FP-value F crit Variety 0.03 2 0.0141 1.2061 0.3034 3.0804 Location 0.033 0.0103 0.8772 0.4554 2.6887 Interaction 0.06 6 0.0097 0.8249 0.55312.1837 Within 1.26 108 0.0117 Total 1.38 119 Anova shows no significantdifferences in frame leaf index at harvest maturity stage betweenVarieties. The average matured leaf index of Caballero (ICE 16171),Serengeti and Desert Classic are 1.06, 1.06 and 1.03, respectively.

TABLE 7 Frame Leaf Area (cm2), calculated by multiplying the leaf lengthby the leaf width Trial NO. Trial1 Trial2 Trial3 Trial4 Trial No. Trial1 Trial 2 Trial 3 Trial 4 Caballero (ICE 16171) 783 676 1085 930 672 840840 910 841 1190 810 870 780 1190 891 840 870 728 957 837 810 812 8101184 756 729 728 736 725 1020 810 924 840 840 754 754 702 1225 957 624Serengeti 870 1020 1188 961 650 1056 841 870 784 1155 810 1120 780 1023899 992 812 957 812 1188 896 1056 960 702 725 1044 840 870 624 930 864784 780 870 784 1155 891 1088 960 980 Desert Classic 1050 952 924 780690 870 1020 990 868 1120 840 729 870 1054 870 1056 783 1056 841 825 8401056 868 924 868 1296 870 930 870 1184 858 1089 899 1295 1088 1147 9301120 899 1258 Anova: Two-Factor With Replication SUMMARY Trial 1 Trial 2Trial 3 Trial 4 Total Caballero (ICE 16171) Count 10 10 10 10 40 Sum7779 9250 8642 8609 34280 Average 777.90 925.00 864.20 860.90 857.00Variance 4181.66 45102.22 11865.29 22200.10 22041.59 Serengeti Count 1010 10 10 40 Sum 7812 10199 8958 9622 36591 Average 781.20 1019.90 895.80962.20 914.78 Variance 8795.96 6732.77 14182.84 25636.18 20850.18 DesertClassic Count 10 10 10 10 40 Sum 8668 11003 9078 9728 38477 Average866.80 1100.30 907.80 972.80 961.93 Variance 8623.96 18343.12 6820.1828341.51 22348.74 Total Count 30 30 30 30 Sum 24259 30452 26678 27959Average 808.63 1015.07 889.27 931.97 Variance 8455.83 27089.79 10550.3426273.07 ANOVA Source of Variation SS df MS F P-value F crit Variety220937.72 2 110468.8583 6.6009 0.0020 3.0804 Location 666615.80 3222205.2667 13.2775 0.0000 2.6887 Interaction 70331.95 6 11721.99170.7004 0.6498 2.1837 Within 1807432.00 108 16735.4815 Total 2765317.47119 Anova shows significant differences in leaf area (cm2) at harvestmaturity stage between Varieties. The average leaf area (cm2) ofCaballero (ICE 16171), Serengeti and Desert Classic are 857.00, 914.78and 961.93, respectively.

TABLE 8 Core Length (cm) at Harvest Maturity Trial NO. Trial 1 Trial 2Trial 3 Trial 4 Caballero (ICE 16171) 2.0 3.0 3.0 5.0 3.0 3.0 2.0 2.03.0 3.0 3.0 3.0 3.0 3.0 2.0 3.0 3.0 3.0 3.0 4.0 3.0 3.0 3.0 4.0 3.0 4.04.0 4.0 2.0 4.0 3.0 3.0 2.0 3.0 3.0 4.0 3.0 3.0 4.0 4.0 Serengeti 3.03.0 3.0 3.0 2.0 3.0 2.0 3.0 2.0 3.0 3.0 2.0 2.0 2.0 2.0 2.0 1.0 3.0 2.03.0 2.0 3.0 2.0 3.0 1.0 3.0 2.0 3.0 2.0 3.0 3.0 2.0 2.0 3.0 3.0 3.0 2.03.0 2.0 2.0 Desert Classic 3.0 2.0 3.0 3.0 2.0 2.0 2.0 3.0 2.0 2.0 2.03.0 3.0 2.0 3.0 4.0 3.0 3.0 2.0 3.0 1.0 2.0 3.0 3.0 2.0 2.0 2.0 3.0 2.03.0 4.0 2.0 2.0 3.0 2.0 2.0 4.0 2.0 3.0 3.0 Anova: Two-Factor WithReplication SUMMARY Trial 1 Trial 2 Trial 3 Trial 4 Total Caballero (ICE16171) Count 10.00 10.00 10.00 10.00 40.00 Sum 27.00 32.00 30.00 36.00125.00 Average 2.70 3.20 3.00 3.60 3.13 Variance 0.23 0.18 0.44 0.710.47 Serengeti Count 10.00 10.00 10.00 10.00 40.00 Sum 19.00 29.00 24.0026.00 98.00 Average 1.90 2.90 2.40 2.60 2.45 Variance 0.32 0.10 0.270.27 0.36 Desert Classic Count 10.00 10.00 10.00 10.00 40.00 Sum 24.0023.00 26.00 29.00 102.00 Average 2.40 2.30 2.60 2.90 2.55 Variance 0.710.23 0.49 0.32 0.46 Total Count 30.00 30.00 30.00 30.00 Sum 70.00 84.0080.00 91.00 Average 2.33 2.80 2.67 3.03 Variance 0.51 0.30 0.44 0.59ANOVA Source of Variation SS df MS F P-value F crit Variety 10.62 2.005.31 14.89 0.00 3.08 Location 7.69 3.00 2.56 7.19 0.00 2.69 Interaction3.98 6.00 0.66 1.86 0.09 2.18 Within 38.50 108.00 0.36 Total 60.79119.00 Anova shows significant differences in core length at harvestmaturity stage between Varieties. The average core length (cm) ofCaballero (ICE 16171), Serengeti and Desert Classic are 3.13, 2.45 and2.55, respectively.

TABLE 9 Core Diameter (cm) at Harvest Maturity Trial NO. Trial 1 Trial 2Trial 3 Trial 4 Caballero (ICE 16171) 3.0 3.0 3.0 3.0 3.0 3.0 3.0 2.03.0 3.0 4.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.03.0 3.0 2.0 3.0 3.0 3.0 2.0 3.0 3.0 4.0 3.0 3.0 3.0 3.0 Serengeti 3.04.0 4.0 3.0 2.0 3.0 3.0 4.0 2.0 3.0 3.0 2.0 2.0 3.0 3.0 3.0 2.0 3.0 3.03.0 2.0 3.0 3.0 3.0 2.0 3.0 3.0 3.0 2.0 3.0 3.0 2.0 2.0 3.0 3.0 3.0 3.03.0 3.0 3.0 Desert Classic 3.0 3.0 3.0 3.0 2.0 3.0 3.0 3.0 2.0 3.0 4.03.0 2.0 3.0 3.0 3.0 3.0 3.0 2.0 3.0 2.0 3.0 3.0 3.0 3.0 3.0 2.0 3.0 2.03.0 3.0 3.0 3.0 3.0 3.0 2.0 3.0 3.0 3.0 3.0 Anova: Two-Factor WithReplication SUMMARY Trial 1 Trial 2 Trial 3 Trial 4 Total Caballero (ICE16171) Count 10 10 10 10 40 Sum 28 30 31 30 119 Average 2.80 3.00 3.103.00 2.98 Variance 0.18 0.00 0.10 0.22 0.13 Serengeti Count 10 10 10 1040 Sum 22 31 31 29 113 Average 2.20 3.10 3.10 2.90 2.83 Variance 0.180.10 0.10 0.32 0.30 Desert Classic Count 10 10 10 10 40 Sum 25 30 29 29113 Average 2.50 3.00 2.90 2.90 2.83 Variance 0.28 0.00 0.32 0.10 0.20Total Count 30 30 30 30 Sum 75 91 91 88 Average 2.50 3.03 3.03 2.93Variance 0.26 0.03 0.17 0.20 ANOVA Source of Variation SS df MS FP-value F crit Variety 0.60 2 0.3000 1.8947 0.1553 3.0804 Location 5.833 1.9417 12.2632 0.0000 2.6887 Interaction 1.60 6 0.2667 1.6842 0.13172.1837 Within 17.10 108 0.1583 Total 25.13 119 Anova shows nosignificant differences in core diameter at harvest maturity stagebetween varieties. The average core diameter (cm) of Caballero (ICE16171), Serengeti and Desert Classic are 2.98, 2.83 and 2.83,respectively.

DEPOSIT INFORMATION

A deposit of the lettuce seed of this invention is maintained byShamrock Seed Company Inc., 3 Harris Place, Salinas, Calif. 93901-4593,USA. In addition, a sample of the lettuce seed of this invention hasbeen deposited with the National Collections of Industrial, Food andMarine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen, Scotland, AB243RY, United Kingdom.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain of the present invention meetsthe criteria set forth in 37 C.F.R. 1.801-1.809, Applicants hereby makethe following statements regarding the deposited lettuce cultivarCABALLERO (deposited as NCIMB Accession No. ______):

1. During the pendency of this application, access to the invention willbe afforded to the Commissioner upon request;2. All restrictions on availability to the public will be irrevocablyremoved upon granting of the patent under conditions specified in 37 CFR1.808;3. The deposit will be maintained in a public repository for a period of30 years or 5 years after the last request or for the effective life ofthe patent, whichever is longer;4. A test of the viability of the biological material at the time ofdeposit will be conducted by the public depository under 37 C.F.R.1.807; and5. The deposit will be replaced if it should ever become unavailable.Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. §122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2,500 seeds of thesame variety with the NCIMB.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

What is claimed is:
 1. A seed of lettuce designated CABALLERO, wherein arepresentative sample of seed of said lettuce has been deposited underNCIMB No. ______.
 2. A lettuce plant, a plant part thereof or a plantcell thereof, produced by growing the seed of claim 1, wherein thelettuce plant or a lettuce plant regenerated from said plant part orplant cell has all of the physiological and morphologicalcharacteristics of lettuce designated CABALLERO listed in Table 1 whengrown under the same environmental conditions.
 3. The lettuce plantpart, or a plant cell thereof of claim 2, wherein the lettuce part isselected from the group consisting of a leaf, a flower, a head and acell.
 4. A lettuce plant, a plant part, or a plant cell thereof, whereinthe plant, or a plant regenerated from the plant part or the plant cellhas all of the physiological and morphological characteristics oflettuce designated CABALLERO listed in Table 1 when grown under the sameenvironmental conditions, wherein a representative sample of seed ofsaid lettuce has been deposited under NCIMB No. ______.
 5. A tissueculture of regenerable cells produced from the plant or plant part ofclaim 2, wherein a plant regenerated from the tissue culture has all ofthe physiological and morphological characteristics of lettuce CABALLEROlisted in Table 1 when grown in the same environmental conditions.
 6. Alettuce plant regenerated from the tissue culture of claim 5, said planthaving all the physiological and morphological characteristics oflettuce CABALLERO hybrid listed in Table 1 when grown under the sameenvironmental conditions wherein a representative sample of seed of saidlettuce plant has been deposited under NCIMB No. ______.
 7. A lettucehead or leaf produced from the plant of claim
 2. 8. A method forharvesting a lettuce head comprising a) growing the lettuce plant ofclaim 2 to produce a lettuce head, and b) harvesting said lettuce head.9. A lettuce head produced by the method of claim
 9. 10. A method forharvesting a lettuce leaf comprising a) growing the lettuce plant ofclaim 2 to produce a lettuce leaf, and b) harvesting said lettuce leaf11. A lettuce leaf produced by the method of claim
 10. 12. A method forproducing a lettuce seed comprising crossing a first parent lettuceplant with a second parent lettuce plant and harvesting the resultantlettuce seed, wherein said first parent lettuce plant and/or secondparent lettuce plant is the lettuce plant of claim
 2. 13. An F1 lettuceseed produced by the method of claim
 12. 14. A method for producing alettuce seed comprising self-pollinating the lettuce plant of claim 2and harvesting the resultant lettuce seed.
 15. A lettuce seed producedby the method of claim
 14. 16. A method of producing a lettuce plantderived from the lettuce CABALLERO, the method comprising (a) crossingthe plant of claim 2 with a second lettuce plant to produce a progenyplant.
 17. The method of claim 16 further comprising the steps of: (b)crossing the progeny plant derived from lettuce CABALLERO with itself ora second lettuce plant to produce a seed of progeny plant of subsequentgeneration; (c) growing the progeny plant of the subsequent generationfrom the seed; (d) crossing the progeny plant of the subsequentgeneration with itself or a second lettuce plant to produce a lettuceplant derived from the lettuce CABALLERO and (e) repeating step b)and/or c) to produce a lettuce plant derived from the lettuce CABALLERO.18. A lettuce plant comprising a single locus conversion and otherwiseessentially all of the characteristics of CABALLERO listed in Table 1when grown under the same environmental conditions, wherein arepresentative sample of seed of said lettuce has been deposited underNCIMB No. ______.
 19. The plant of claim 19 wherein the single locusconversion confers said plant with herbicide resistance.
 20. The plantof claim 18 wherein the single locus conversion is an artificiallymutated gene or nucleotide sequence.
 21. The plant of claim 18 whereinthe single locus conversion is a gene that has been modified through theuse of a New Breeding Technique.
 22. A method for producing nucleicacids, the method comprising isolating nucleic acids from the plant ofclaim 2, or a plant part, or a plant cell thereof.
 23. A method forproducing a second lettuce plant, the method comprising applying plantbreeding techniques to the plant or plant part of claim 2 to produce thesecond lettuce plant.
 24. A method of introducing a desired trait intolettuce CABALLERO comprising: (a) crossing a lettuce CABALLERO plantgrown from lettuce CABALLERO seed, wherein a representative sample ofseed has been deposited under NCIMB No. ______, with another lettuceplant that comprises a desired trait to produce F1 progeny plants; (b)selecting one or more progeny plants that have the desired trait toproduce selected progeny plants; (c) crossing the selected progenyplants with the lettuce CABALLERO plants to produce backcross progenyplants; (d) selecting for backcross progeny plants that have the desiredtrait and all of the physiological and morphological characteristics oflettuce CABALLERO listed in Table 1 when grown in the same environmentalconditions to produce selected backcross progeny plants; and (e)repeating steps (c) and (d) three or more times in succession to produceselected fourth or higher backcross progeny plants that comprise thedesired trait and all of the physiological and morphologicalcharacteristics of lettuce CABALLERO listed in Table 1 when grown in thesame environmental conditions.